CD40 ligand fusion protein vaccine

ABSTRACT

Provided are methods of generating an immune response to any of various antigens including foreign antigens such as infectious agent antigens. In general, the method comprises administering an expression vector encoding a transcription unit encoding a secretable fusion protein, the fusion protein containing the foreign antigen and CD40 ligand and also administering the encoded fusion protein. In another approach, an immune response to the foreign antigen is elicited using the encoded fusion protein without administering the vector. The invention methods may be used to immunize an individual against an infectious agent such as influenza virus. Methods of obtaining an immune response in older individuals also is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/593,458, filed on Nov. 6, 2006, which claims priority and the benefitof U.S. Provisional Application No. 60/734,136 filed Nov. 7, 2005, U.S.Provisional Application No. 60/755,885 filed Jan. 4, 2006, U.S.Provisional Application No. 60/789,270 filed Apr. 4, 2006, U.S.Provisional Application No. 60/793,206 filed Apr. 19, 2006, and U.S.Provisional Application No. 60/853,184 filed Oct. 20, 2006, thedisclosures of which are all hereby incorporated herein by reference intheir entireties.

STATEMENT OF GOVERNMENT SPONSORED RESEARCH

This invention was made with government support under ARMY/MRMC GrantNos. DAMD17-03-1-0554 and DAMD17-99-1-9457, and Grant No. R43 CA108051.The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of vaccines. Inparticular, the present invention relates to the use of fusion proteinsof CD40 ligand and an antigen in developing immunity to foreign proteinsor infectious agents.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Influenza is an acute contagious illness often characterized byinflammation of the respiratory tract, fever, chills, muscular pain,prostration and maliase and is caused by the Orthomyxoviridae family ofinfluenza viruses. Infection can cause mild to severe illness, and attimes can lead to death.

Influenza viruses are classified into three types: Types A, B, and C.Type A influenzas have been responsible for pandemics, spreading over awide geographic area and affecting a large proportion of the population.Type A influenza viruses are known to infect many animals includingbirds and mammals (e.g., humans, dogs, horses, cattle, sheep, pigs andseals). In contrast, type B influenzas tend usually to infect onlyhumans. Type A and B influenzas are responsible for the increasedflu-related illnesses, hospitalizations and deaths that occur each year.Type C influenzas tend to be the least worrisome. Infection in humansmay cause mild respiratory distress or no symptoms at all.

Type A influenza viruses are further classified by strain. The strainname is determined by identifying differences between two antigenicproteins, hemagglutinin (“HA”) and neuraminidase (“NA”), both present onthe viral surface. Rapid alterations in the sequence of these twoproteins (termed “antigenic shift”) are mainly responsible for theyearly changes in immunogenicity and the requirement for new vaccineseach year. Examples of antigenic shift and the concomitant strain changeare clear: between 1918 and 1957, the H1N1 strain was dominant; between1957 and 1968, the H2N2 strain was dominant; then, since 1968, the H3N2virus dominated. Most recently, in 1997, the H5N1 strain of avianinfluenza was shown to infect humans. In addition to antigenic shift,the antigenic properties of influenza viruses can also change moreslowly via “antigenic drift,” the slow, gradual process of viralevolution. Antigenic variation (drift) of HA sequences is noted, forexample, in human trials with the escape of viral strains from vaccineinduced immunity.

One cause of the rapid sequence changes associated with “antigenicshift” is the formation of reassortant viral strains. The pig can beinfected by both human and avian influenza strains, and an exchange ofRNA stands between human and avian strains can occur within the pig.These “reassortant” viruses may infect the human species causing ayearly epidemic of influenza. Thus, the rapid changes in the HA and NAproteins resulting from antigenic shift render old influenza viralvaccines ineffective.

Hemagglutinin (“HA”), one of the proteins affected by antigenic shift,is an antigenic glycoprotein found on the surface of influenza viruses.HA functions to secure the virus to the target cells by binding toN-acetyl neuraminic acid or sialic acid on host cell receptors. HA iscomposed of two subunits, HA1 and HA2. HA2 is the viral membraneanchoring domain, while HA1 is responsible for binding host cellreceptors. As noted above, HA is highly mutable and variations, mainlyin HA1, are a key source of viral antigen variability conferring theability to evade the immune system. There are currently between 16 and20 different HA varieties known, H1, H2 and H3 have been the dominanthuman influenza subtypes, while the H5 and H7 subtypes have beenprevalent for avian species.

Neuraminidase (“NA”) is also presented on the viral surface andfunctions to catalyze the removal of terminal sialic acid residues ofglycosyl groups, thus destroying potential receptors for hemagglutinin.It is probable that neuraminidase is required to prevent viralaggregation and to promote more efficient spreading of the virus fromcell to cell. The neuraminidase protein sequence is also highlyvariable; there are currently nine different neuraminidase varietiesknown. Accordingly, changes in this protein sequences also play a rolein antigenic shift.

Another viral protein present on the viral influenza surface is theMatrix protein 2 (“M2”), an ion channel protein that selectively allowsprotons to enter the virus. After the virus enters a cell, an influx ofprotons is key in the removal of the viral protein coat. The M2 proteinis homotetramer comprised of three domains: a 23 amino-acid regionpresent on the outside of the virus (extra cellular domain), a 54 aminoacid region that is inside the virus (cytoplasmic domain) and a 19amino-acid transmembrane domain. M2 is expressed at low levels on theviral surface but is present at high levels on influenza infected cells.The M2 protein sequence is stable compared to hemagglutinin orneuraminidase. In fact, the 23-amino acid extra cellular domain of M2 iswell conserved in many known influenza strains (some exceptions includeA/PR/8/34, A/Brevig and Mission/1/8). Although this protein is notimmunogenic normally, it has been shown that chimeric molecules madefrom the extra cellular domain of the M2 and “adjuvant proteins” such asthe hepatitis B core protein induce a potent immune response (Virology2005 337:149-161; Infection and Immunity 2002 70:6860-70),

The intranasal administration of the M2HB core particle, along withadjuvants (such as a detoxified enterotoxin adjuvant), protected 2-4month old BalbC mice from challenge with human influenza virus (Virology2005 337:149-161). M2, which is important in determining host range (J.Virology 1999 73:3366-3374), is present at such low levels in the virusthat antibodies are not generated. HA has been the target for vaccinessince the antibodies to H5 have been shown to prevent influenza viralinfection.

A commercial human vaccine against the H5N1 strain of influenza has notyet been developed, although the H5 strain of avian influenza virus wasseen to infect human directly in 1997; 6/18 infected people died. Sincethat time, outbreaks have occurred in 2003 in Hong Kong (2 deaths in 3cases), and in Vietnam/Thailand in 2004 (28 deaths in 39 infected cases(Kash J C et al., Journal of Virology 78: 9499-9511 (2004);Apisarnthanarak A, et al., Emerg. Infect. Dis. 10 (2004)). In 2005,79/150 H5N1 infected human beings died. Over 99% of the sequences of theinfecting H5N1 virsus are avian, suggesting direct transmission from thepoultry to human beings (Science 2001 293:1840-1842; J. Virology 200074:1443-1450). When the avian influenza viruses acquire the capabilityof directly jumping from birds to humans, there is a potential for apandemic. For this to occur, the virus must be able to be transmitted inaerosols from person to person. A transition from avian:human tohuman:human transmission resulting in a pandemic was documented for thefirst time in 1918 in an H1N1 strain. The result was more than 600,000deaths in the USA and 40 million worldwide (J. Virology 2004 78:9499-9511). Statistics show that most of the deaths were restricted toyounger individuals living in crowded conditions (soldiers involved inWorld War I).

The H5N1 strain has been reported to infect humans (J. Virology 200074:1443-1450). It is estimated that if this virus acquires thecapability of spreading from human to human, there impact in the USAwill be over 200,000 deaths, over 700,000 hospitalizations, over 40million outpatient visits, and an economic impact of over 100 billiondollars (Infect. Dis. 1999 5:659-671). The recent apparent trend forincreased reporting deaths of individuals believed infected with theavian flu virus has created concern among governments around the world(J. Virology 2004 78:9499-9511; J. Emere, Infect. Dis. 2004 10; Virology2003 208:270-278; Eur. J. Biochem. 1999 260:166-175).

The testing of vaccine efficacy for the H5N1 avian flu has been carriedout in mice and ferrets, but the virulence of the various human strainsin ferrets is closer to that seen in humans (J. Virology 200579:2191-2198). The pathogenicity of the various strains has beencorrelated with the HA protein structure (Id). After the 1997 cases inHong Kong, two strategies for vaccines against the H5N1 strains weretested. First, it was discovered that a subunit H5 vaccine did notappear to be immunogenic in humans (J. Infect. Dis. 2005 191:1213-1215;Vaccine 2003 21:1687-1693). However, the addition of MF59 adjuvantincreased the antibody response (PNAS USA 2005 102:12915-12920). In asecond approach, a multivalent vaccine (H3N1 and H5N1) was used but thisproved equally ineffective (Virology 1999 73:2094-2098).

Recently, an HA DNA vaccine was shown to protect mice from the H5N1strain(clintrials.gov/ct/gui/sho/NCT00110279;jsessionid=743259FCCOA680603EA),and there is currently a clinical trial to evaluate the immune responseto H9N2 avian flu in humans (Vaccine 2002 20:1099-1105). The H9N2 studyinvolves a cold-adapted resorted attenuated viral vaccine which isadministered by intramuscular injection. It is hoped that this studywill provide insight into an H5N1 immune response. Additionally, anintramuscularly administered vaccine from baculovirus expressing H5 HAwas tested in 147 adults. A 23% antibody response was observed after asingle injection and 52% response after two injections (Lancet 2004357:1937-1943).

The elderly are especially at risk with respect to influenza infection,and vaccination against influenza is recommended for older individualsto prevent the potentially deadly complications of infection such aspneumonia or bronchitis. One cause of increased risk in the elderly isthe decrease in function of the immune system with age. For example,there is a decrease in the number of naïve, antigen unexposed CD4 andCD8 T cells. Additionally, the ratio of the naïve to memory CD8/CD4cells decreases as the chronological age increases. Further, CD4 cellsbecome impaired, acquiring both quantitiative and functional defects,such as diminished levels of the CD40 ligand (CD40L) on the surface ofCD4 cells as well as a temporal retardation of the rate at which CD40ligand (CD40L) is expressed on the surface of the CD4 cells followingactivation. Accordingly, the amount of antibody that an elderly systemis able to generate will be lower following infection or conventionalvaccination.

Testing has shown that current methods of vaccination are, at best, onlymoderately effective. Usually, three strains of the human influenzavirus are grown up in eggs, purified and then chemically inactivated.Using the induction of neutralizing antibodies in the vaccinatedindividuals as an endpoint for response, the response to the vaccine isin the 65-70% range (Lancet 2004 357:1937-1943). The response is 4-foldless in individuals vaccinated after age 55.

Vaccines have been described that include an expression vector encodinga fusion protein that includes an antigen fused to CD40 ligand. See,e.g., U.S. Patent Application Publication US 2005-0226888 (applicationSer. No. 11/009,533) titled “Methods for Generating Immunity toAntigen,” filed Dec. 10, 2004.

SUMMARY OF THE INVENTION

According to the present invention there are provided methods ofgenerating an immune response to a fusion protein having CD40 ligand anda foreign antigen. In some embodiments the fusion protein isadministered as an expression vector containing DNA encoding the fusionprotein. In other embodiments, the fusion protein is directlyadministered as a protein. Vaccination regimens wherein vector andprotein are administered are also provided.

Thus, in a first aspect, there are provided new vaccines for protectingagainst infection by influenza viruses. An immune response to aninfluenza antigen is achieved by administering an expression vectorencoding a secretable fusion protein which includes an influenza antigenand CD40 ligand.

The influenza antigen may be any influenza antigen to which an immuneresponse may be generated in an individual or animal. In preferredembodiments, the influenza antigen is an mammalian influenza virusantigen (such as a human influenza antigen) or an avian influenza virusantigen. In further embodiments, the influenza antigen may be acombination of mammalian and avian influenza virus antigens, such as acombination of human and avian influenza virus antigens. The influenzaantigen is preferably an influenza viral protein, or fragment thereof,which comprises at least one antigenic determinant.

In a preferred embodiment, the influenza antigen of the fusion proteinis the matrix protein 2 (“M2”) ion channel protein. The M2 protein,which is a tetrameric 23 amino acid long type III transmembrane proteininvolved in tropism of the virus, is barely detectable on the influenzavirus but is expressed at high levels on influenza virus infected cells.In contrast to the HA protein, the M2 has a stable sequence from year toyear among different influenza strains.

It is believed that the invariant nature of the M2 antigen when fused toCD40 ligand in the present vaccine design, will provide a “universal”influenza vaccine that, unlike current vaccines, will be effectiveagainst different strains of influenza resulting from antigenic drift.This is especially important in that there may not be time to prepare avaccine against a known unique HA antigen for the avian influenza virusonce a pandemic occurs, as there is each year for the human influenzareassorted virus.

In one embodiment, the M2 antigen of the fusion protein lacks all orsubstantially all of the M2 transmembrane domain. Preferably, the M2antigen of the fusion protein includes all of the extracellular domainof M2 or at least an antigenic fragment of the extracellular domain ofM2.

In other embodiments, the influenza antigen may be a chimeric influenzaantigen having at least one antigenic determinant from at least twoviral influenza proteins. Such different antigens may be variants of thesame antigen (e.g. hemagglutinins from different virus strains, e.g.from H5N1, H2N2, H3N2 or H1N1 strains) or a chimeric influenza antigenhaving at least one antigenic determinant from at least two differentviral influenza proteins (e.g. HA and M2).

In some embodiments, the influenza antigen may be a viral protein orfragment thereof. For example, the viral protein or fragment may be ahemagglutinin (“HA”), a neuraminidase (“NA”), an M2 or any combinationof HA, NA or M2. The influenza antigen may comprise: an extracellulardomain or domain fragment of the viral antigen (e.g., HA, NA or M2); acytoplasmic domain or domain fragment; or a combination of extracellularand cytoplasmic sequences from the same protein. In some embodiments,the influenza antigen may be a protein chimera having any immunogeniccombination of different influenza proteins (e.g., any combination ofhemagglutinin, neuraminidase or M2 proteins or fragments thereof). Theinfluenza protein May be from any strain for which protection is sought.In some embodiments, the influenza antigen may be from an H5N1, H3N2,H1N1 or H2N2 influenza strain. In some embodiments the influenza antigenis a chimeric of HA and M2 proteins or fragments thereof. In otherembodiments, the influenza antigen is a chimeric of HA and M2extracellular domain regions from one or more influenza strains (e.g.,from H5N1, H3N2, H1N1 or H2N2).

As used herein an “antigen” is any foreign material that is specificallybound by the combining site of an antibody or by the combining site of aT cell antigen receptor. Antigens may also be immunogens if they areable to trigger an immune response, or haptens if not.

As used herein, “antigenic determinant” refers to a single antigenicsite or epitope on a complex antigenic molecule or particle, a minimalportion of a molecule that interacts with an antibody or T cellreceptor. Antigenic determinants may be linear or discontinuous.

The fusion protein encoding an influenza antigen may be administeredbefore, concurrently or after administration of the vector. Preferably,the fusion protein is administered after the vector. The sequence of theinfluenza antigen encoded by the vector and that present in the fusionprotein may be identical or may be different. If different, the twopreferably have at least one antigenic determinant in common.

In one approach, the sequence encoding the influenza antigen in thefusion protein transcription unit is 5′ to sequence encoding the CD40ligand. In another approach, the sequence encoding the CD40 ligand inthe fusion protein transcription unit o sequence encoding the influenzaantigen. In a preferred embodiment, the CD40 ligand lacks all or aportion of its transmembrane domain.

In another aspect, the invention provides methods of immunizing anindividual against infection by an influenza virus. The method includesadministering an expression vector which includes a transcription unitencoding a secretable fusion protein that contains an influenza antigenassociated and CD40 ligand. A fusion protein that encodes an influenzaantigen associated with the virus and CD40 ligand may also beadministered before, concurrently or after administration of the vector.Preferably, the fusion protein is administered after the vector. In thisapproach, the influenza antigen which is encoded by the vector is chosento cross-react with the influenza virus for which protection is beingsought. The immune response generated by this approach is both cellmediated and humoral (i.e. antibody response). The antibody response canneutralize infectious influenza virus.

In preferred embodiments, the expression vector may be a viralexpression vector or a non-viral expression vector; the expressionvector may be an adenoviral vector; the vector may be advantageouslyadministered subcutaneously; the vector may be administered on asubsequent occasion(s) to increase the immune response; a signalsequence may be placed upstream of the fusion protein for secretion ofthe fusion protein; immunity against the antigen may he long lasting andinvolve generation of cytotoxic CD8⁺ cells against antigen expressingcells and the production of antibody to the antigen; the transcriptionunit may include sequence that encodes a linker between the antigen andthe CD40 ligand; suitable linkers may vary in length and composition;the expression vector may include a human cytomegaloviruspromoter/enhancer for controlling transcription of the transcriptionunit; and the CD40 ligand may be a human CD40 ligand.

In a further aspect, the invention provides methods of effectivelyimmunizing older individuals by administering an expression vectorencoding a secretable fusion protein which includes an antigen and CD40ligand. Older individuals which may realize an improved immune responseusing the methods of the invention as compared to other immunizationmethods are at least 50 years of age, or even at least 55 years of ageor even yet at least 60 years of age.

The achievement of improved immune responses with the invention methodsas compared to other immunization approaches are applicable to any ofvarious antigens which are fused to CD40 ligand. Such antigen includecancer antigens (tumor associated or tumor specific) or infectious agentantigens. Infectious agent antigens may be bacterial, viral, fungal,protozoan, and the like. Viral antigens may be an influenza antigen asdescribed above. The viral antigen may be from a human papilloma virus.The viral antigen may be the E6 or E7 protein of human papilloma virus.

It is believed that traditional vaccination is poorly effective in olderindividuals because the expression level of the CD40 ligand (CD40L) onthe surface of CD4 cells following activation is greatly reduced inolder individuals as compared to young, healthy individuals. On thebasis of this functional CD4 defect alone, it is believed that thelevels of antibodies that will be made following the conventionalvaccination with recombinant antigen and inactivated viral particlevaccines, even with adjuvants, will be lower in older individuals ascompared to younger individuals. It is further believed that poor immuneresponses in older individuals may be due in part to the diminishedexpression of CD40L by CD4 cells. The administration of an expressionvector in accordance with the methods of the invention is believed toresult in in vivo activation and antigen loading of dendritic cells(DCs) following release of the fusion protein from vector infectedcells. The loading of DC with the CD40 ligand from the fusion proteinbypasses the CD4 cell of older individuals which are deficient in CD40L.It has been found that two sc injections of this vaccine vector inducesan immune response that last for over a year and which is independent ofCD4 cells.

In yet another aspect, the invention provides a vector and the fusionprotein encoded thereby along the lines described herein for generatingan immune response in an individual against an influenza antigen.

In still another aspect, there is provided a method of increasing theimmune responsiveness of an individual having CD4 T cells exhibitingreduced levels of CD40 ligand as compared to young, healthy individualsto vaccination against a cancer antigen or an infectious agent antigen.The method is accomplished by administering to the individual aneffective amount of an expression vector having a transcription unitencoding a secretable fusion protein, having a cancer or infectiousagent antigen and CD40 ligand.

In a further aspect, there are provided methods of immunizingindividuals above the age of 50 against foreign antigens. Such methodsare particularly suited for generating immunity to influenza in olderindividuals, which represent the majority group for which influenza A aswell as avian influenza protection is needed.

In another aspect, the invention provides a method of generating animmune response in an individual against a foreign antigen. This methodis accomplished by administering to the individual an effective amountof a fusion protein without administering an expression vector encodingthe fusion protein, wherein the administering is repeated, and whereinthe fusion protein contains the antigen and CD40 ligand. The foreignantigen may be from an infectious agent such as influenza. The foreignantigen of the fusion protein may be glycosylated or non-glycosylated.The immune response generated-.by this approach may be both cellmediated and humoral (i.e. antibody response). The antibody response canneutralize infectious influenza virus.

Abbreviations used herein include “Ad” (adenoviral); “sig” (signalsequence and “ecd” (extra cellular domain); “sc” (subcutaneous).

These and other embodiments are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an amino sequence of an M2 protein from an H5N1 influenzavirus (GenBank Accession No. AF036358, SEQ ID NO:1).

FIG. 2 shows an amino sequence of an HA protein (without the N-terminal16 amino acid signal peptide) from an H5N1 influenza virus (GenBankAccession No. AF036356, SEQ ID NO:2).

FIG. 3 shows an amino sequence of an M2 protein from an H3N2 influenzavirus (GenBank Accession No. AAA43276, SEQ ID NO:3)

FIG. 4 shows an amino sequence of an HA protein from an H3N2 influenzavirus (GenBank Accession No. V01085, SEQ ID NO:4).

FIG. 5 shows a nucleotide sequences encoding A) a fragment of an HAprotein from an H5N1 influenza virus, and B) a fragment of an M2 proteinfrom an H5N1 influenza virus. (SEQ ID NOs:5 and 6, respectively)

FIG. 6 depicts the growth of E7-positive TC-1 and E7-negative EL-4 cellsin Ad-sig-E7/ecdCD40L vector vaccinated mice. E7-positive TC-1 cells(diamonds); E7-negative EL-4 cells (squares).

FIG. 7 depicts growth of E7-positive TC-1 cells versus E7-negative EL-4cells in mice injected with splenic T lymphocytes from donor micevaccinated with Ad-sig-E7/ecdCD40L vector.

FIG. 8 depicts percent survival days after injection of test mice. Thetest mice were first injected with CD8+ cells (black diamonds), CD4cells (triangles) or no cells (squares) from Ad-sig-E7/ecdCD40L vectorvaccinated mice. Seven days later the test mice were injected withE7-positive Tc-1 cells.

FIG. 9 depicts the level of E7-specific T cell response after injectionof different vector constructs.

FIG. 10 shows the levels of splenocytes producing interferon-gamma andIL4 in Ad-sig-E7lecdCD40L vector vaccinated and unvaccinated young andold mice.

FIG. 11 shows the percentage of antigen specific T cells in the CD8population antigen positive tumor of old animals unimmunized (control)and immunized with Ad-sig-E7/ecdCD40L vector.

FIG. 12 shows the percentage of CD4 and CD8 T cells infiltrating tumornodules in old animals vaccinated with Ad-sig-E7/ecdCD40L vector and inyoung animals unvaccinated.

FIG. 13 depicts cytotoxic T cell activity at various effector to targetratios induced by Ad-sig-E7/ecdCD40L vector vaccination of young and oldmice.

FIG. 14 depicts the suppression of E7 tumor growth in old and youngAd-sig-E7/ecdCD40L vector vaccinated mice.

FIG. 15 depicts the percentage of E7 tumor free mice after vaccinationwith the Ad-sig-E7/ecdCD40L vector and protein boost in old and youngmice.

FIG. 16 shows the percentage of FoxP3 regulatory CD4+ cells among tumorinfiltrating lymphocytes in the tumor before (control) and aftervaccination (old) of old (18 month) mice with Ad-sig-E7/ecdCD40L vector.

FIG. 17 shows the percentage of effector CD8+ cells in tumor nodules ofrH2n.Tg (i.e., HER2 transgenic) mice injected withAd-sig-rH2N/ecdΔCtΔTmCD40L vector.

FIG. 18 depicts the level of H5HA-specific CD8 T cells in the spleen ofAd-sig-H5HA/ecdCD40L vaccinated mice compared to unvaccinated mice viaELISA spot assay interferon gamma detection.

FIG. 19 depicts an exemplary amino acid sequence of neuraminidase H5N1(FIG. 19A) and the encoding nucleotide sequence (FIG. 19B) of GenBank IDNo. g2865377 (SEQ ID NO:7 and SEQ ID NO:8, respectively).

FIG. 20 depicts an exemplary amino acid sequence of an HA from H5N1 (SEQID NO:9; GenBank ID g2865380). Underlined are residues reported to beinvolved in receptor binding.

FIG. 21 depicts an exemplary precursor HA molecule from strain H3N2(GenBank Accession No. V01086; SEQ ID NO:10). Underlined and italicizedsequence is involved in receptor binding or represents an epitopeagainst which neutralizing antibodies have been generated.

FIG. 22 depicts an exemplary amino acid sequence of neuraminidase frominfluenza A virus strain A/Memphis/31/98 (H3N2), GenBank ID g30385699(SEQ ID NO: 11). Residues reported to be mutated in known escape mutantsare shown underlined in bold. The extracellular domain is shaded.

FIG. 23 depicts the level of CD8 T cells in a preparation of splenocytesfrom Ad-sig-H5HA/ecdCD40L vaccinated old and young mice compared tounvaccinated mice, as determined by via ELISpot assay interferon gammadetection.

FIG. 24 demonstrates the presence of antibodies against HA in serum ofold and young mice following vaccination with Ad-sig-H5HA/ecdCD40Lvector followed by three protein boosts of HA/ecdCD40L fusion protein.

FIG. 25 depicts the level of M2-specific CD8 T cells in a preparation ofsplenocytes from Ad-sig-H5M2/ecdCD40L vaccinated old and young micecompared to unvaccinated mice, as determined by via ELISpot assayinterferon gamma detection.

FIG. 26 demonstrates the presence of antibodies against M2 in serum ofold and young mice following vaccination with Ad-sig-H5M2/ecdCD40Lvector followed by two protein boosts of M2/ecdCD40L fusion protein.

FIG. 27 depicts the level of E7-specific CD8 T cells in a preparation ofsplenocytes from 2 month old mice (“Young”) vaccinated according to theV, VP, VPP, or VPPP vaccination regimens wherein “V” means to administerthe Ad-sig-E7/ecdC40L and “P” means to administer the E7/ecdCD40Lprotein.

FIG. 28 depicts the level of E7-specific antibody in serum from 2 monthold mice vaccinated according to the V, VP, VPP, or VPPP vaccinationregimens wherein “V” means to administer the Ad-sig-E7/ecdC40L and “P”means to administer the E7/ecdCD40L protein.

FIG. 29 depicts the level of E7-specific CD8 T cells in a preparation ofsplenocytes from 18 month old mice (“Old”) vaccinated according to theV, VP, VPP, or VPPP vaccination regimens wherein “V” means to administerthe Ad-sig-E7/ecdC40L and “P” means to administer the E7/ecdCD40Lprotein.

FIG. 30 depicts the level of E7-specific antibody in serum from 18 monthold mice (“Old”) vaccinated according to the V, VP, VPP, or VPPPvaccination regimens wherein “V” means to administer theAd-sig-E7/ecdC40L and “P” means to administer the E7/ecdCD40L protein.

FIG. 31 demonstrates the presence of antibodies against HA in serum ofmice following a vaccination by three administrations of HA/ecdCD40Lfusion protein.

FIG. 32 demonstrates the presence of antibodies against M2 in serum ofmice following vaccination by three administrations of M2/ecdCD40Lfusion protein.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one aspect of the invention, a method is provided forgenerating an immune response against an influenza antigen using anexpression vector and/or the fusion protein encoded thereby. The vectorincludes a transcription unit encoding a secretable fusion proteincontaining the influenza antigen and CD40 ligand. In a preferredembodiment, the transcription unit includes from the amino terminus, asecretory signal sequence, the influenza antigen, a linker and asecretable form of CD40 ligand. In preferred embodiments, the secretableform of CD40 ligand lacks all or substantially all of its transmembranedomain.

In a preferred approach, the individual is first administered the vectoron one or more occasions to generate a primary immune response. A fusionprotein having the influenza antigen and CD40 ligand protein is alsoadministered in an effective amount after administration of vector toboost the immune response to the antigen above that obtained with vectoradministration alone.

The term “in an effective amount” in reference to administering thefusion protein is an amount that generates an increased immune responseover that obtained using the expression vector alone. A time intervalbetween administrations is generally required for optimal results. Anincrease in the immune response may be measured as an increase in T cellactivity or antibody production. Generally, at least one week betweenvector administration and protein boosting is effective although ashorter interval may be possible. An effective spacing betweenadministrations may be from 1 week to 12 weeks or even longer. Multipleboosts may be given which may be separated by from 1-12 weeks or evenlonger periods of time.

The use of the fusion protein to boost the immune response avoids havingto repetitively administer the expression vector which might generatehypersensitivity to multiple injections. The antigen portion of thefusion protein is preferably the fusion protein which is encoded by thetranscription unit of the expression vector used in the initialadministration. However, the antigen portion of the fusion protein maydiffer from the encoded antigen provided that there is at least oneshared antigenic determinant or epitope common to the antigen of theexpression vector and that of the fusion protein used for boosting.

The fusion protein may be produced in a variety of cell systems. Incertain embodiments, the antigen is desired to be glycosylated. In theseembodiments, if the foreign protein contains a glycosylation signal, acell system that produces glycosylated proteins can be used, such as aeukaryotic cell, preferably a mammalian cell, Exemplary cell systemsinclude CHO cells, COS cells, and MDCK cells. Avian cells may also beused when avian-specific glycosylation is desired. In some embodiments,glycosylation of the foreign protein portion of the fusion protein maybe avoided by producing the fusion protein synthetically or by using anon-glycosylation system such as a bacterial expression system.

The fusion protein may be prepared in a mammalian cell line system,which is complementary to the vector. For example, in the case ofadenovirus, the cell line system can be 293 cells that contain the EarlyRegion 1 (E1) gene and can support the propagation of the E1-substitutedrecombinant adenoviruses. When the adenoviral vectors infect theproduction cells, the viral vectors will propagate themselves followingthe viral replication cycles. However, the gene of interest that iscarried by the viral vector in the expression cassette will expressduring the viral propagation process. This can be utilized forpreparation of the fusion protein encoded by the vector in the samesystem for production of the vector. The production of both the vectorand the fusion protein will take place simultaneously in the productionsystem. The vector and protein thus produced can be further isolated andpurified via different processes.

The fusion protein may also be prepared in non-mammalian cells, such asbacterial cells. For example, cDNA encoding the fusion protein can besubcloned into a vector such as pTriEx Hygro (Novagen, Inc.) andtransfected into E. coli (e.g., Rosetta cells from Novagen, Inc.) wherethe fusion protein is produced. Other non-mammalian cells include yeast,algae, insect, and plant cells.

The fusion protein may be administered parenterally, such asintravascularly, intravenously, intraarterially, intramuscularly,subcutaneously, or the like. Administration can also be orally, nasally,rectally, transdermally or inhalationally via an aerosol. The proteinboost may be administered as a bolus, or slowly infused. The proteinboost is preferably administered subcutaneously.

The fusion protein boost may be formulated with an adjuvant to enhancethe resulting immune response. As used herein, the term “adjuvant” meansa chemical that, when administered with the vaccine, enhances the immuneresponse to the vaccine. An adjuvant is distinguished from a carrierprotein in that the adjuvant is not chemically coupled to the immunogenor the antigen. Adjuvants are well known in the art and include, forexample, mineral oil emulsions (U.S. Pat. No. 4,608,251, supra) such asFreund's complete or Freund's incomplete adjuvant (Freund, Adv. Tuberc.Res. 7:130 (1956); Calbiochem, San Diego Calif.), aluminum salts,especially aluminum hydroxide or ALHYDROGEL (approved for use in humansby the U.S. Food and Drug Administration), muramyl dipeptide (MDP) andits analogs such as [Thr¹]-MDP (Byers and Allison, Vaccine 5:223(1987)), monophosphoryl lipid A (Johnson et al., Rev. Infect. Dis.9:S512 (1987)), and the like,

The fusion protein can be administered in a microencapsulated or amacroencapsulated form using methods well known in the art. Fusionprotein can be encapsulated, for example, into liposomes (see, forexample, Garcon and Six, J. Immunol. 146:3697 (1991)), into the innercapsid protein of bovine rotavirus (Redmond et al., Mol. Immunol. 28:269(1991)) into immune stimulating molecules (ISCOMS) composed of saponinssuch as Quil A (Morein et al., Nature 308:457 (1984)); Morein et al., inImmunological Adjuvants and Vaccines (G. Gregoriadis al. eds.) pp.153-162, Plenum Press, NY (1987)) or into controlled-releasebiodegradable microspheres composed, for example, of lactide-glycolidecopolymers (O'Hagan et al., Immunology 73:239 (1991); O'Hagan et al.,Vaccine 11:149 (1993)).

The fusion protein also can be adsorbed to.the surface of lipidmicrospheres containing squalene or squalane emulsions prepared with aPLURONIC block-copolymer such as L-121 and stabilized with a detergentsuch as TWEEN 80 (see Allison and Byers, Vaccines: New Approaches toImmunological Problems (R. Ellis ed.) pp. 431-449, Butterworth-Hinemann,Stoneman N.Y. (1992)). A microencapsulated or a macroencapsulated fusionprotein can also include an adjuvant.

The fusion protein also may be conjugated to a carrier or foreignmolecule such as a carrier protein that is foreign to the individual tobe administered the protein boost. Foreign proteins that activate theimmune response and can be conjugated to a fusion protein as describedherein include proteins or other molecules with molecular weights of atleast about 20,000 Daltons, preferably at least about 40,000 Daltons andmore preferably at least about 60,000 Daltons. Carrier proteins usefulin the present invention include, for example, GST, hemocyanins such asfrom the keyhole limpet, serum albumin or cationized serum albumin,thyroglobulin, ovalbumin, various toxoid proteins such a tetanus toxoidor diphtheria toxoid, immunoglobulins, heat shock proteins, and thelike.

Methods to chemically couple one protein to another (carrier) proteinare well known in the art arid include, for example, conjugation by awater soluble carbodiimide such as1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride, conjugationby a homobifunctional cross-linker having, for example, NHS ester groupsor sulfo-NHS ester analogs, conjugation by a heterobifunctionalcross-linker having, for example, and NHS ester and a maleimide groupsuch as sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate and, conjugation with gluteraldehyde (see, forexample, Hermanson, Bioconjugate Techniques, Academic Press, San Diego,Calif. (1996)); see, also, U.S. Pat. Nos. 4,608,251 and 4,161,519).

The term “vector” which contains a transcription unit (aka. “expressionvector”) as used herein refers to viral and non-viral expression vectorsthat when administered in vivo can enter target cells and express anencoded protein. Viral vectors suitable for delivery in vivo andexpression of an exogenous protein are well known and include adenoviralvectors, adeno-associated viral vectors, retroviral vectors, herpessimplex viral vectors, and the like. Viral vectors are preferably madereplication defective in normal cells. See U.S. Pat. Nos. 6,669,942;6,566,128; 6,794,188; 6,110,744; 6,133,029.

As used herein, the term “cells” is used expansively to encompass anyliving cells such as mammalian cells, plant cells, eukaryotic cells,prokaryotic cells, and the like.

The term “adenoviral expression vector” as used herein, refers to anyvector from an adenovirus that includes exogenous DNA inserted into itsgenome which encodes a polypeptide. The vector must be capable ofreplicating and being packaged when any deficient essential genes areprovided in trans. An adenoviral vector desirably contains at least aportion of each terminal repeat required to support the replication ofthe viral DNA, preferably at least about 90% of the full FUR sequence,and the DNA required to encapsidate the genome into a viral capsid. Manysuitable adenoviral vectors have been described in the art. See U.S.Pat. Nos. 6,440,944 and 6,040,174 (replication defective E1 deletedvectors and specialized packaging cell lines). A preferred adenoviralexpression vector is one that is replication defective in normal cells.

“Adenoviral expression vectors” may include vectors that have beenmodified to better target and infect specific cell types (e.g.,fibroblasts and dendritic cells), or that have been modified to avoidneutralization by pre-existing, high-titer antibodies, such as theantibodies circulating in humans against Ad5 and Ad2.

Adeno-associated viruses represent a class of small, single-stranded DNAviruses that can insert their genetic material at a specific site onchromosome 19. The preparation and use of adeno-associated viral vectorsfor gene delivery is described in U.S. Pat. No. 5,658,785.

Non-viral vectors for gene delivery comprise various types of expressionvectors (e.g., plasmids) which are combined with lipids, proteins andother molecules (or combinations of thereof) in order to protect the DNAof the vector during delivery. Fusigenic non-viral particles can beconstructed by combining viral fusion proteins with expression vectorsas described. Kaneda, Curr Drug Targets (2003) 4(8):599-602.Reconstituted HVJ (hemagglutinating virus of Japan; Sendaivirus)-liposomes can be used to deliver expression vectors or thevectors may be incorporated directly into inactivated HVJ particleswithout liposomes. See Kaneda, Curr Drug Targets (2003) 4(8):599-602.DMRIE/DOPE lipid mixture are useful a vehicle for non-viral expressionvectors. See U.S. Pat. No. 6,147,055. Polycation-DNA complexes also maybe used as a non-viral gene delivery vehicle. See Thomas et al., ApplMicrobial Biotechnol (2003) 62(1):27-34.

The term “transcription unit” as it is used herein in connection with anexpression vector means a stretch of DNA that is transcribed as asingle, continuous mRNA strand by RNA polymerase, and includes thesignals for initiation and termination of transcription. For example, inone embodiment, a transcription unit of the invention includes nucleicacid that encodes from 5′ to 3,′ a secretory signal sequence, aninfluenza antigen and CD40 ligand. The transcription unit is in operablelinkage with transcriptional and/or translational expression controlelements such as a promoter and optionally any upstream or downstreamenhancer element(s). A useful promoter/enhancer is the cytomegalovirus(CMV) immediate-early promoter/enhancer. See U.S. Pat. Nos. 5,849,522and 6,218,140.

The term “secretory signal sequence” (aka. “signal sequence,” “signalpeptide,” leader sequence,” or leader peptide”) as used herein refers toa short peptide sequence, generally hydrophobic in charter, includingabout 20 to 30 amino acids which is synthesized at the N-terminus of apolypeptide and directs the polypeptide to the endoplasmic reticulum.The secretory signal sequence is generally cleaved upon translocation ofthe polypeptide into the endoplasmic reticulum. Eukaryotic secretorysignal sequences are preferred for directing secretion of the exogenousgene product of the expression vector. A variety of suitable suchsequences are well known in the art and include the secretory signalsequence of human growth hormone, immunoglobulin kappa chain, and thelike. In some embodiments the endogenous tumor antigen signal sequencealso may be used to direct secretion.

The term “antigen” as used herein refers broadly to any antigen to whicha human, mammal, bird or other animal can generate an immune response.“Antigen” as used herein refers broadly to a molecule that contains atleast one antigenic determinant to which the immune response may bedirected. The immune response may be cell mediated or humoral or both.

As is well known in the art, an antigen may be protein in nature,carbohydrate in nature, lipid in nature, or nucleic acid in nature, orcombinations of these biomolecules. As is well known in the art, anantigen may be native, recombinant or synthetic. For example, an antigenmay include non-natural molecules such as polymers and the like.Antigens include both self antigens and non-self antigens. “Self”antigens include antigens encoded by the host's genome. Self antigensinclude those variant sequences that arise through natural recombinationevents in the host genome. For example, the variable regions ofimmunoglobulin genes recombine in many combinations to produce a largediversity in immunoglobulins. Other self antigens may include proteinsthat are overexpressed or underexpressed in disease states such ascancer. For example, various mucin isoforms are overexpressed in certaincancer types.

“Foreign” antigens, as used herein refer to non-self antigens. Foreignantigens may be the products of or encoded by the genome of otherorganisms. For example, a foreign antigen to a mammal can be an antigenencoded by an infectious agent such as a microbe. Infectious agentantigens may be bacterial, viral, fungal, protozoan, and the like.

The term “influenza virus” as used herein refers to any of the influenzavirus Types A, B and C that can infect mammals or birds. “Influenza” asused herein refers to an acute contagious influenza virus infection thatis generally characterized by fever, chills, muscular pain, prostrationand that generally involves the respiratory system with symptoms such asinflammation of the respiratory tract.

The term “hemagglutinin” (“HA”) is a major glycoprotein that comprisesover 80% of the envelope proteins present in the influenza virusparticle. Heinagglutinin binds to sialic acid-containing receptors onthe cell surface, bringing about the attachment of the virus particle tothe cell. Hemagglutinin also is responsible for penetration of the virusinto the cell cytoplasm by mediating the fusion of the membrane of theendocytosed virus particle with the endosomal membrane. Low pH inendosomes induce an irreversible conformational change in HA2, releasingthe fusion hydrophobic peptide.

The term “hemagglutinin” refers to the full length protein and fragmentsthereof which share at least one antigenic determinant with full lengthhemagglutinin. “Hemagglutinin” as used herein may be native, recombinantor synthetic and may be post-transitionally modified such as byglycosylation and/or palmitoylation. There are currently at leastsixteen different known subtypes of hemagglutinin characterizedantigenicly, termed H1 through H16.

Hemagglutinin is synthesized as a precursor of about 566 amino acids. Anexemplary amino sequence of an HA from an H5N1 virus is shown in FIG. 2and an HA from an H3N2 virus is shown in FIG. 4. The sequence of varioushemagglutinins are found in protein and nucleotide databases such asSwissProt and GenBank. See, e.g., Swiss Prot accession nos. P03437,P03441, P19694, P19695, P12581, P07976, P07977, P09345, GenBankaccession no. AF036356 (H5N1 virus); and V01085 (H3N2 virus). Thesedatabases notate the various functional domains of the HA protein. Forexample, SwissProt accession no. P03437 discloses a sequence of an H3hemagglutinin. This molecule is synthesized as a 566 aa precursor. Aminoacids 1-16 represent a 16 aa signal sequence; 17-530 represents a 514 aaextracellular domain; 531-551 represents a 21 aa transmembrane domain;and 552-566 represents a 15 aa cytoplasmic domain. Nucleotide sequenceencoding the extracellular domain of an HA is shown in FIG. 5a . The 566amino acid precursor is exported to the cell membrane of influenza viruswhere it gets cleaved into the HA1 and HA2 subunits. The HA 1 subunitrepresents 17-344 of the HA precursor while the HA2 subunit represents345-566 of the HA precursor. The HA molecule has a large extracellulardomain of about 500 aa. A posttranslational cleavage by host-derivedenzymes generates 2 polypeptides that remain linked by a disulfide bond.Thus, the larger N-terminal fragment HA1 which includes about 320-330 aaforms a membrane-distal globular domain (or head) of about 170 aminoacids that contains the receptor-binding site and most antigenicdeterminants recognized by virus-neutralizing antibodies. The smallerC-terminal portion HA2 which includes about 180 aa (excludingtransmembrane and cytoplasmic domain) forms a stem-like structure thatanchors the globular domain to the cellular or viral membrane. The hingeregion is located between the stem and globular domain.

Sixteen HA subtypes have been currently identified among influenza Aviruses; three of these (H1, H2, H3) have been associated with classicinfluenza isolates, and 3 (H5, H7, H9) have been associated with recentsporadic human isolates. Influenza B viruses possess only 1 HA subtype.Thus, the sequence for each HA and the positions of the variousfunctional domains of the HA can differ and are easily determined by oneskilled in the art.

In some embodiments, the antigen is the entire extracellular domain ofHA. Alternatively, smaller regions of the extracellular domain may serveas the antigen for invention vaccines. In choosing an antigen, one ofskill in the art would recognize that one could select an antigen thatis recognized by MHC class II molecules to elicit an antibody(“humoral”) response or an antigen that is recognized by MHC class Imolecules to illicit a cytotoxic T cell (“cellular”) response. Inpreferred embodiments, a region of the viral protein is chosen thatencompasses antigens recognized by both MHC I and MHC II. Such a regionmay be a contiguous stretch of amino acids from the native protein ormay include discontinuous regions linked together.

In addition, antigenic regions of the HA molecule have been identifiedthrough the production of antibodies, some of which are neutralizing.The literature identifies four sites (A through D) on the HA moleculethat tend to elicit antibody responses. Sites A, B, and D can be foundon the head portion of the HA molecule, while site C is on the hinge(Nature 1981 289:373-8).

In one example, fragments of HA from the H5N1 strain of influenza areused as the influenza antigen. The sequence set forth in SEQ ID NO:9represents an exemplary amino acid sequence of a precursor HA moleculefrom strain H5N1 (GenBank ID g2865380). The extracellular domain of thissequence is from about amino acid 17 to 530. In some embodiments, theentire extracellular domain is used as the influenza antigen. In otherembodiments, one or more fragments of the extracellular domain of HA areused. Preferred fragments of the extracellular domain of SEQ ID NO:9 areshown below. Further, there are a number of epitopes predicted to bindMHC class I or MHC class II molecules within this region. Exemplarypredicted MHC class I epitopes and MHC class II epitopes are underlinedand italicized, respectively, in the sequences below.

(SEQ ID NO: 12) NHFEKIQIIPKSSWSNHDASSGVSSACPYLGRSSFFRNVV WLIKKNSAY PTIKRSYNNTNQEDLLVLWGVHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPEIATRPKVNGQSGRMEFFWTILKPNDAINFESN; (SEQ ID NO: 13)NHFEKIQIIPKSSWSNHDASSGVSSACPYLGRS SFFRNVVWLI KKNSAYP TIKRSYNNTNQ; (SEQID NO: 14) IQIIPKSSWSNHDASSGVSSACPYLGRSSFFRNV VWLIKKNSAY PTIKRS Y; (SEQID NO: 15) KSSWSNHDASSGVSSACPYLGRSSFFRNVVWLIKKNSAYPT; (SEQ ID NO: 16)TLNQRLVPEIATRPKVNGQSGRMEFFWTILKPNDAINFESN (SEQ ID NO: 17)LVPEIATRPKVNGQSGRMEFFWTILKPNDAI; or (SEQ ID NO: 18)ATRPKVNGQSGRMEFFWTILK.

Additional antigen sequences are possible and may be identified with anyof a number of computer programs known in the art for that purpose. Forexample, “SYFPEITHI is a publicly available database for MHC ligands andpeptide motifs is supported by DFG-Sonderforschungsbereich, 510 and theEuropean Union: EU BIOMED CT95-1627, BIOTECH CT95-0263, and EUQLQ-CT-1999-00713(www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm).

In further embodiments the focus of the influenza antigen one coulddesign an antigen that is dominated by portions of the HA receptorbinding site which elicit neutralizing antibodies (i.e., antibodies thatprevent host infection by, for example, the blocking of HA binding tothe HA receptor). For example, neutralizing antibodies to H5N1 (GenBankID g2865380, SEQ ID NO:9) have been described in the literature to reactmainly with (amino acid residues Y91, W149, E186, and L190 and sequencesSGVSS (SEQ ID NO:19, corresponding to amino acid residues 129-133) andNGQSG (SEQ ID NO:20, corresponding to amino acid residues 220-224)(Science 279:393-6, 1998, FIG. 1). These residues are shown in FIG. 20as underlined residues. Thus, an influenza antigen may include mainlythe two sites SGVSS (SEQ ID NO:19) and NGQSG (SEQ ID NO: 20). As isknown in the art, the receptor binding site can vary from HA molecule toHA molecule from different strains and even within a strain.

An exemplary “influenza antigen” includes a protein fragmentrepresenting the receptor binding region of HA from an H5N1 virus,repressing amino acids 134-205 (see FIG. 2).

Combinations of two or more of the above fragments i nay be used as theantigen. Such fragments may be joined by a linker or may be immediatelyadjacent to each other. One example of a combination of two fragments is

(SEQ ID NO: 21) KSSWSNHDASSGVSSACPYLGRSSFFRNVVWLIKKNSAYPTATRPKVNGQSGRMEFFWTILK,which represents SEQ ID NO:15 immediately adjacent and upstream of SEQID NO:18. One skilled in the art would recognize that these fragmentscould be joined with SEQ ID NO:18 upstream of SEQ ID NO:15 as well.

In another example, fragments of HA from the H3N2 strain of influenzaare the influenza antigen. The sequence set forth in SEQ ID NO:10 andshown in FIG. 21 represents an exemplary amino acid sequence of aprecursor HA molecule from strain H3N2 (GenBank ID Accession No.V01086). The extracellular domain of this sequence is from about aminoacid 17 to 530. The extracellular domain of this sequence is from aboutamino acid 17 to 530. In some embodiments, the entire extracellulardomain is used as the antigen in invention vaccines. In otherembodiments, one or more fragments of the extracellular domain of HA areused. Preferred fragments of the extracellular domain of SEQ ID NO:10are shown below.

Preferred fragments of the extracellular domain of SEQ ID NO:10 areshown below.

(SEQ ID NO: 22) TITNDQIEVTNATELVQSSSTGKICNNPHRILDGINCTLIDALLGDPHCDGFQNEKWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFINEGFNWTGVTQNGGSSACKRGPDSGFFSRLNWLYKSGSTYPVQNVTMPNNDNSDKLYIWGVHHPSTDKEQTNLYVQASGKVTVSTKRSQQTIIPNVGSRPWVRGLSSRISIYWTIVKPGDILVINSNGNLIAPRGYFK.

SEQ ID NO:22 comprises antigenic regions recognized by MHC class Imolecules. The following sequences represent exemplary MHC class Iepitopes.

(SEQ ID NO: 23) TITNDQIEV; (SEQ ID NO: 24) ILDGINCTLIDA; (SEQ ID NO: 25)LFVERSKAF; (SEQ ID NO: 26) PYDVPDYASLRSLVASSG; (SEQ ID NO: 27) WLYKSGSTY(SEQ ID NO: 28) NNDNSDKLY; and (SEQ ID NO: 29) STDKEQTNLY.

SEQ ID NO:22 comprises antigenic regions recognized by MHC class Hmolecules. The following sequences represent exemplary MHC class IIepitopes.

(SEQ ID NO: 30) TELVQSSSTGKICNN; (SEQ ID NO: 31) PHRILDGINCTLIDA; and(SEQ ID NO: 32) VPDYASLRSLVASSG.

Further preferred fragments include those residues reported to beinvolved in receptor binding of a precursor HA molecule from strain H3N2(SEQ ID NO:10). Such residues are shown in FIG. 21 as underlinedresidues. Thus, preferred fragments include, for example, EFINEG (SEQ IDNO:33). A further preferred fragment is KAFSNCYPYDVPDY (SEQ ID NO:34)which has been shown to be an epitope against which neutralizingantibodies have been generated (Int Arch Allergy Immunol 2002127:245-50).

One of skill in the art would recognize one could join two or more ofthe above sequences to form an antigen. Further one could extend thesequence of any of the fragments by, for example, 5 amino acids or morepreferably, 10 amino acids, on either end or both ends.

The term neuraminidase (“NA”) as used herein refers to a glycoproteinfound on the surface of influenza viruses. Neuraminidase catalyses theremoval of terminal sialic acid residues of glycosyl groups, therebydestroying potential receptors for HA. It is believed that neuraminidaseensures the efficient spread of the virus by dissociating the maturevirions from the neuraminic acid containing glycoproteins. Thus,NA-specific antibodies inhibit the release of newly formed virus frominfected host cells and thereby limit the spread and shedding of virusduring infection.

The term “neuraminidase” refers to the full length protein and fragmentsthereof which share at least one antigenic determinant with full lengthneuraminidase. “Neuraminidase” as used herein may be native, recombinantor synthetic and may be post-translationally modified such as byglycosylation. There are currently at least nine neuraminidasesub-types.

Neuraminidase is synthesized as a precursor of about 469 amino acids.The sequence of various NA are found in protein and nucleotide databasessuch as Swiss-Prot and GenBank. See, e.g., Swiss-Prot accession nosP06818, P26143, Q9Q0U7, P06820, GenBank accession no. AF028708 (H5N1virus), and AB124658 (H3N2 virus). These databases annotate the variousfunctional domains of the NA protein. For example, SwissProt accessionno. P06818 discloses a sequence of an N2 Neuraminidase from influenza Avirus (strain A/Bangkok/1/79). This molecule is synthesized as a 469 aaprecursor. Amino acids 1-6 represent a 6 aa cytoplasmic domain; 7-35represent a 29 aa transmembrane domain; and 36-469 represents a 434 aaextracellular domain. The extracellular domain consists of a 55 aahypervariable stalk region (amino acid residues 36 through 90) and a 379aa head region (amino acid residues 91 through 469). The sequence foreach NA and the positions of the various functional domains of the NAcan differ and are easily determined by one skilled in the art.

In some embodiments, the influenza antigen is from the neuraminidaseprotein In preferred embodiments, the entire extracellular domain ofneuraminidase is used as the influenza antigen. Alternatively, fragmentsof the extracellular domain may also be selected as the influenzaantigen. In such fragments, one of skill in the art can use any of anumber of computer software programs for choosing antibody epitopes orantigens for MHC class I or MHC class II molecules, known in the art anddiscussed above. In preferred embodiments the antigen is focused aroundregions of the neuraminidase protein known in the art to be antigenicand, more particularly, regions which include residues of known escapemutants. “Escape mutants” as used herein are proteins which have amutation in an antigenic region such that an antibody that previouslybound to that region will no longer bind. In this way, such mutantsescape or evade an immune response. In other embodiments, one or morefragments of the extracellular domain of NA are used.

In one example, the influenza antigen is neuraminidase from influenzavirus A/Memphis/31/98, H3N2. An exemplary sequence NA from H3N2 is setforth in SEQ ID NO:11 (GenBank ID No, g30385699) and is shown in FIG.22. The extracellular domain of SEQ ID NO:11 is indicated in FIG. 22(shaded region) and is the influenza antigen in some embodiments.Furthermore, several escape mutants have been described in theliterature. For example, Gulati and coworkers describe escape mutantswith mutations at amino acid positions 198, 199, 220, and 221(underlined and in bold in FIG. 22) (J Virol 2002 76(23):12274-80).Preferred fragments of SEQ ID NO:11 are shown below.

QCKITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTLNNRHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCVTGHDENATASFIYDGRLVDSIGSWSKKILRTQESECVCINGTCTVVMTDGSASGRADTKILFIEEGKIVHISPLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINVKDYSIVSSYVCSGLVGDTPRKNDSSSSSHCLNPNNEEGGHGVKGWAFDDGNDVWMGRTISEKFRSGYETFKVIEGWSKPNSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETEVWWTSNSIVVFCGTSGTYGTGSWPDGADINLMPI (SEQ ID NO: 35, corresponds to the headregion of NA); AWLHVCVTGHDENATASFIYDGRLVDSIGSWSKKILRTQESECV (SEQ ID NO:36); CVTGHDENATASFIYDGRLVDSIGSWSKKILRTQ (SEQ ID NO: 37); andDENATASFIYDGRLVDSIGSWSKK (SEQ ID NO: 38).

The term Matrix protein 2 (“M2”) as used herein refers to an integralmembrane protein expressed on the surface of the influenza virus and theinfected cells. M2 is expressed at high levels of influenza virusinfected cells, and compared to hemagglutinin and neuraminidase has astable sequence from year to year among different influenza strains.Although this protein is not typically immunogenic, it has been shownthat chimeric molecules made from the extracellular domain of M2 and“adjuvant proteins” such as the hepatitis B core protein induce a potentimmune response. (Virology 2005 337:149-161; Infection and Immunity 200270:6860-70).

M2 is synthesized as a precursor of about 109 amino acids. An exemplaryamino sequence of an M2 from an H5N1 virus is shown in FIG. 1 and an M2from an H3N2 virus is shown in FIG. 3. The sequence of various M2 arefound in protein and nucleotide databases such as SwissProt and GenBank.See, e.g., Swiss Prot accession nos. P13881, P13882, P03493, Q80DN6,P08383, P0C0X4, P21430, P03491, GenBank accession no. AF036358 (H5N1virus); and AAA43276 (H3N2 virus). These databases annotate the variousfunctional domains of the M2 protein. For example, SwissProt accessionno. P21430 (influenza A virus) discloses a sequence of an M2. Thismolecule is synthesized as a 97 aa precursor. Amino acids 1-22 representa 22 aa extracellular domain; 23-43 represents a 21 aa transmembranedomain; and 44-97 represents an 54 aa cytoplasmic domain. Nucleotidesequence encoding the extracellular domain of an M2 is shown in FIG. 5b. The sequence for each M2 and the positions of the various functionaldomains of the M2 can differ and are easily determined by one skilled inthe art. For example, M2 from Influenza B virus has a very short 4 aaextracellular domain.

The term “influenza antigen” as used herein refers to any part, portionor region of an influenza virus protein that can elicit an immuneresponse in mammals or birds. Influenza antigens can be native,recombinant or synthetic. An “influenza antigen” may be include epitopesderived from a single viral type or strain or from multiple viral typesor strains. An “influenza antigen” may be a fission protein of epitopesfrom the same or different viral types or strains, An “influenzaantigen” as used herein may include the HA, NA or M2 protein, orfragments thereof containing one or more epitopes; an “influenzaantigen” may contain the entire extracellular domain of HA, NA or M2,the cytoplasmic domain of HA, NA or M2 and/or any immunogeniccombination of these proteins or domains.

An exemplary extracellular domain of M2 from Hong Kong/485/97(H5N1)matrix protein 2 (GenBank Accession No:AJ278648)

(SEQ ID NO: 39) MSLLTEVDTLTRNGWGCRCSDSSD,which is encoded by the nucleotide sequence

(SEQ ID NO: 40) 5′-ATG AGC CTT CTA ACC GAG GTT GAC ACG CTT ACC AGA AACGGA TGG GGG TGC AGA TGC AGC GAT TCA AGT GAT- 3′.

A further example of an M2 antigen Influenza A virus (A/NewYork/522/1997(H3N2) CY006508) is

(SEQ ID NO: 41) 5′-ATGAGCC TTCTAACCGA GGTCGAAACACC TATCAGAAACGA ATGGGGGT GCAGATGCAA CGATTCAAGT GAC-3′.The above sequence encodes the following sequence, corresponding toamino acids 1-24 of the M2 protein,

(SEQ ID NO: 42) MSLLTEVETPIRNEWGCRCNDSSD

Another example of an influenza antigen is a chimeric protein having asegment of an HA and a segment of an M2 protein (e.g. from an H5N1 orH3N2 virus). In one example of a chimeric influenza antigen is thefollowing sequence which encodes an HA-M2 chimera from H5N1.

(SEQ ID NO: 43) 5′-AAAAGTTCTTGGTCCAATCATGATGCCTCATCAGGGGTGAGCTCAGCATGTCCATACCTTGGGAGGTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTGCATACCCAACAGCTACTAGACCCAAAGTAAACGGGCAAAGTGGAAGAATGGAGTTCTTCTGGACAATTTTAAAG GATATC ATGA GCCTTCTAACCGAGGTTGAC ACGCTTACCAGAAACGGATGGG GGTGCAGATG CAGCGATTCAAGTGAT-3′.

In the above exemplary sequence the HA and M2 fragments are joined by anoptional 2 amino acid linker (nucleotides encoding the linker areunderlined in the sequence above). The HA fragment precedes the linkerand the M2 fragment follows the linker. One of skill in the art wouldrecognize that the order of these fragments could be reversed with theM2 fragment preceding the HA fragment. An exemplary nucleotide sequenceencoding such a fragment is below.

(SEQ ID NO: 44) 5′-ATGAGCCTTCTAACCGAGGTTGACACGCTTACCAGAAACGGATGGGGGTGCAGATGCAGCGATTCAAGTGATAAAAGTTCTTGGTCCAATCATGATGCCTCATCAGGGGTGAGCTCAGCATGTCCATACCTTGGGAGGTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTGCATACCCAACAGCTACTAGACCCAAAGTAAACGGGCAAAGTGGAAGAATGGAGTTCTTCTGGA CAATTTTAAAG-3′.

Another example of a chimeric influenza antigen includes fragments of HAand M2 (underlined) from H3N2 and is as follows,

(SEQ ID NO: 45) TITNDQIEVTNATELNQSSSTGKICNNPHRILDGINCTLIDALLGDPHCDGFQNEKWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFINEGFNWTGVTQNGGSSACKRGPDSGFFSRLNWLYKSGSTYPVQNVTMPNNDNSDKLYIWGVHHPSTDKEQTNLYVQASGKVTVSTKRSQQTIIPNVGSRPWVRGLSSRISIYWTIVKPGDILVINSNGNLIAPRGYFKMSLLTEVETPIRNEWGCR CNDSSD.

Other chimeras can be constructed from fragments of neuraminidase and M2or HA. For example, the following sequence consists of the head regionof neuraminidase and a the extracellular domain of M2 (underlined), bothfrom H3N2.

(SEQ ID NO: 46) QCKITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTLNNRHSNDTVHDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCVTGHDENATASFIYDGRLVDSIGSWSKKILRTQESECVCINGTCTVVMTDGSASGRADTKILFIEEGKIVHISPLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINVKDYSIVSSYVCSGLVGDTPRKNDSSSSSHCLNPNNEEGGHGVKGWAFDDGNDVWMGRTISEKFRSGYETFKVIEGWSKPNSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKQETEVWWTSNSIVVFCGTSGTYGTGSWPDGADINLMPIMSLLTEVETPIRNEWGCRCND SSD.

One of skill in the art would recognize that fragments from differentproteins of different strains of influenza viruses may be combined toform an antigen. In some embodiments. M2 comprises one of the twofragments. In the following example, a fragment of HA from H5N1 iscombined with an M2 fragment (underlined) from H3N2,

(SEQ ID NO: 47) KSSWSNHDASSGVSSACPYLGRSSFFRNVVWLIKKNSAYPTATRPKVNGQSGRMEFFWTILK MSLLTEVETPIRNEWGCRCNDSSD.

As used herein, an “influenza antigen-CD40L” means an influenza proteinlinked to CD40L. For example, this may be an H3N2 or H5N1 HA antigenlinked to CD40L; an H3N2 or H5N1 HA-M2 chimeric antigen linked to CD40L;or an H3N2 or H5N1 M2 linked to CD40L. Although the above examples listH3N2 and H5N1, an “influenza antigen” as used herein refers to antigensexpressed by an influenza virus and includes proteins or fragments fromother Type A, B or C influenza viruses.

A secretable form of an antigen is one that lacks all or substantiallyall of its transmembrane domain, if present in the mature protein. Forexample, in the case of a hemagglutinin, the transmembrane domain, ifpresent, is generally about 19-21 amino acids in length and functions toanchor the hemagglutinin or a fragment of the hemagglutinin in the cellmembrane. For example, in the case of the hemagglutinin disclosed inSwissProt accession no. P03437, a secretable form of this hemagglutininin which the transmembrane domain has been deleted is missing residues531-551 of the precursor protein. A hemagglutinin missing substantiallyall of the transmembrane domain is one where the domain comprises 6residues or less of sequence at one end of the transmembrane domain,more preferably less than about 4 residues of sequence at one end of thetransmembrane domain, even more preferably less than about 2 residues ofsequence on one end of the transmembrane domain, and most preferably 1residue or less on one end of the transmembrane domain. In a preferredembodiment, the vaccine vector transcription unit encodes a secretableform of a hemagglutinin lacking the entire transmembrane domain. Ahemagglutinin that lacks substantially all of the transmembrane domainrendering the hemagglutinin secretable is one that contains no more thansix residues of sequence on one end of the domain. The extracellulardomain of a human HA is denoted herein as “ecdHA.”

It should be understood that a hemagglutinin, neuraminidase or M2protein which lacks a functional transmembrane domain (i.e., is insecretable form) may still include all or a portion of the cytoplasmicdomain.

A source of DNA encoding the various hemagglutinins, neuraminidases orM2 antigens may be obtained from hemagglutinins, neuraminidases or M2expressing cell lines using a commercial cDNA synthesis kit andamplification using a suitable pair of PCR primers that can be designedfrom the published DNA sequences. Hemagglutinin, neuraminidase or M2encoding DNA also may be obtained by amplification from RNA or cDNAobtained or prepared from infected human or other animal tissues orcells, or from viral isolates. For DNA segments that are not that large,the DNA may be synthesized using an automated oligonucleotidesynthesizer.

The term “linker” as used herein with respect to the transcription unitof the expression vector refers to one or more amino acid residuesbetween the carboxy terminal end of the antigen and the amino terminalend of CD40 ligand. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. (See e.g. Arai et al., design of the linkers whicheffectively separate domains of a bifunctional fusion protein. ProteinEngineering, Vol. 14, No. 8, 529-532, August 2001). The linker isgenerally from about 3 to about 15 amino acids long, more preferablyabout 5 to about 10 amino acids long, however, longer or shorter linkersmay be used or the linker may be dispensed with entirely. Longer linkersmay be up to about 50 amino acids, or up to about 100 amino acids. Alinker of about 30 residues is preferred when the M2 or hemagglutininantigen is N-terminal to the CD40 ligand. One example of a linkerwell-known in the art is a 15 amino acid linker consisting of threerepeats of four glycines and a serine (i.e., [Gly₄Ser]₃).

The term “CD40 ligand” (CD40L) as used herein refers to a full length orportion of the molecule known also as CD154 or TNFS. CD40L is a type IImembrane polypeptide having a cytoplasmic domain at its N-terminus, atransmembrane region and then an extracellular domain at its C-terminus.Unless otherwise indicated the full length CD40L is designated herein as“CD40L,” “wtCD40L” or “wtTmCD40L.” The form of CD40L in which thecytoplasmic domain has been deleted is designated herein as “ΔCtCD40L.”The form of CD40L where the transmembrane domain has been deleteddesignated herein as “ΔTmCD40L.” The form of CD40L where both thecytoplasmic and transmembrane domains have been deleted is designatedherein as “ΔCtΔTmCD40L” or “ecdCD40L.” The nucleotide and amino acidsequence of CD40L from mouse and human is well known in the art and canbe found, for example, in U.S. Pat. No. 5,962,406 (Armitage et al.).Also included within the meaning of CD40 ligand are variations in thesequence including conservative amino acid changes and the like which donot alter the ability of the ligand to elicit an immune response to amucin in conjunction the fusion protein of the invention.

Murine CD40L (mCD40L) is 260 amino acids in length. The cytoplasmic (Ct)domain of mCD40L extends approximately from position 1-22, thetransmembrane domain extends approximately from position 23-46, whilethe extracellular domain extends approximately from position 47-260.

Human CD40L (hCD40L) is 261 amino acids in length. The cytoplasmicdomain of hCD40L extends approximately from position 1-22, thetransmembrane domain extends approximately from position 23-46, whilethe extracellular domain extends approximately from position 47-261,

The phrase “CD40 ligand is missing all or substantially all of thetransmembrane domain rendering CD40 ligand secretable” as used hereinrefers to a recombinant form of CD40 ligand that can be secreted from acell. The transmembrane domain of CD40L which contains about 24 aminoacids in length, functions to anchor CD40 ligand in the cell membrane.CD40L from which all of the transmembrane domain has been deleted isCD40 ligand lacking residues 23-46. CD40 ligand missing substantiallyall of the transmembrane domain is one that comprises 6 residues or lessof the transmembrane domain, more preferably less than about 4 residuesof transmembrane domain sequence, even more preferably less than about 2residues of transmembrane domain sequence and most preferably 1 residueresidues from the transmembrane domain sequence. Any transmembranesequence that is present from the CD40L may be at one end of the domainor may constitute transmembrane domain sequence contributed by bothends. Thus, a CD40L that lacks substantially all of the transmembranedomain rendering the CD40L secretable is one that retains no more thansix residues of sequence of the transmembrane domain. Such as CD40Lwould contain, in addition to the extracellular domain and optionallythe cytoplasmic domain, no more than amino acids 41-46 or 23-28, or acombination totaling 6 residues or less from both ends of thetransmembrane domain of CD40L. In a preferred embodiment, the vaccinevector transcription unit encodes a secretable form of CD40 containingless than 10% of the transmembrane domain. More preferably, CD40Lcontains no transmembrane domain.

It should be understood that a CD40L which lacks a functionaltransmembrane domain may still include all or a portion of thecytoplasmic domain. Likewise, a CD40L which lacks a functionaltransmembrane domain may include all or a substantial portion of theextracellular domain.

Expression vectors encoding a secretable fusion protein that includes aninfluenza antigen and CD40 ligand can be constructed in accordance withmethods known in the art. See, e.g., U.S. Patent Application PublicationUS 2005-0226888 (application Ser. No. 11/009,533) titled “Methods forGenerating Immunity to Antigen,” filed Dec. 10, 2004, which isincorporated herein in its entirety including the drawings.

As used herein, an expression vector and fusion protein boost isadministered as a vaccine to induce immunity to an influenza antigen.The expression vector and protein boost may be formulated as appropriatewith a suitable pharmaceutically acceptable carrier. Accordingly, thevectors or protein boost may be used in the manufacture of a medicamentor pharmaceutical composition. Expression vectors and the fusion proteinmay be formulated as solutions or lyophilized powders for parenteraladministration. Powders may be reconstituted by addition of a suitablediluent or other pharmaceutically acceptable carrier prior to use.Liquid formulations may be buffered, isotonic, aqueous solutions.Powders also may be sprayed in dry form. Examples of suitable diluentsare normal isotonic saline solution, standard 5% dextrose in water, orbuffered sodium or ammonium acetate solution. Such formulations areespecially suitable for parenteral administration, but may also be usedfor oral administration or contained in a metered dose inhaler ornebulizer for insufflation. It may be desirable to add excipients suchas polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,polyethylene glycol, mannitol, sodium chloride, sodium citrate, and thelike.

Alternately, expression vectors and the fusion protein may be preparedfor oral administration. Pharmaceutically acceptable solid or liquidcarriers may be added to enhance or stabilize the composition, or tofacilitate preparation of the vectors. Solid carriers include starch,lactose, calcium sulfate dihydrate, terra alba, magnesium stearate orstearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriersinclude syrup, peanut oil, olive oil, saline and water. The carrier mayalso include a sustained release material such as glyceryl monostearateor glyceryl distearate, alone or with a wax. The amount of solid carriervaries but, preferably, will be between about 20 mg to about 1 g perdosage unit. When a liquid carrier is used, the preparation may be inthe form of a syrup, elixir, emulsion, or an aqueous or non-aqueoussuspension.

Expression vectors and the fusion protein may be formulated to includeother medically useful drugs or biological agents. The vectors also maybe administered in conjunction with the administration of other drugs orbiological agents useful for the disease or condition that the inventioncompounds are directed.

As employed herein, the phrase “an effective amount,” refers to a dosesufficient to provide concentrations high enough to generate (orcontribute to the generation of) an immune response in the recipientthereof. The specific effective dose level for any particular subjectwill depend upon a variety of factors including the disorder beingtreated, the severity of the disorder, the activity of the specificcompound, the route of administration, the rate of clearance of theviral vectors, the duration of treatment, the drugs used in combinationor coincident with the viral vectors, the age, body weight, sex., diet,and general health of the subject, and like factors well known in themedical arts and sciences. Various general considerations taken intoaccount in determining the “therapeutically effective amount” are knownto those of skill in the art and are described, e.g., in Gilman et al.,eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics,8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Co., Easton, Pa., 1990. For administration ofvectors, the range of particles per administration typically if fromabout 1×10⁷ to 1×10¹¹, more preferably 1×10⁸ to 5×10¹⁰, and even morepreferably 5×10⁸ to 2×10¹⁰. A vector can be administered parenterally,such as intravascularly, intravenously, intraarterially,intramuscularly, subcutaneously, or the like, Administration can also beorally, nasally, rectally, transdermally or inhalationally via anaerosol. The vectors may be administered as a bolus, or slowly infused.The vector is preferably administered subcutaneously.

As described herein, vectors encoding influenza antigens can induce aprotective cellular and humoral immunity against such antigens,including those to which tolerance had developed. Although not wishingto be bound by any theory, it is believed that the invention vaccinesgenerate upon administration a continual local release of the fusionprotein composed of the secretable form of the antigen linked to asecretory form of CD40 ligand. As demonstrated herein this facilitatesDCs maturation, promoting the development of effective antigen-specificimmunity. It is also demonstrated herein that the secretable fusionprotein encoding the extracellular domain of H5N1 HA and the murineCD40L lacking a transmembrane and cytoplasmic domain (i.e.H5HA-ΔCtΔTmCD40L) produced from an adenoviral vector dramaticallyincreased the number of CD8 T cells responsive to H5HA. Although notwishing to be bound by any theory, it is believed that subcutaneousinjection of the Ad-sig-HSHA-ΔCtΔTmCD40L vector elicits a strong HAspecific CD8⁺ T cell-mediated immunity.

The immunity generated against the antigens using the invention methodsis long lasting. As used herein, the term long lasting means thatimmunity elicited by the antigen encoded by the vector can bedemonstrated for up to 6 months from the last administration, morepreferably for up to 8 months, more preferably for up to one year, morepreferably up to 1.5 years, and more preferably for at least two years.

In one embodiment, immunity to an influenza hemagglutinin can begenerated by producing a fusion protein that comprises part of theextracellular domain of the hemagglutinin fused to the amino-terminalend of the CI)40 ligand from which the transmembrane and cytoplasmicdomains were deleted. Construction of such vector is disclosed in theExamples. As was observed herein, subcutaneous administration of thisadenoviral vector hemagglutinin vaccine induced a CD8⁺ cytotoxic T celllymphocyte dependent systemic immune response.

Although not wishing to be bound by any theory, it is believed that thecells infected in the vicinity of the site of subcutaneous injection ofthe vector release the antigen/CD40 ligand secretory which is taken upby antigen presenting cells (e.g. DCs) in the vicinity of the infectedcells. The internalized influenza antigen would be digested in theproteosome with the resultant influenza antigen peptides trafficking tothe endoplasmic reticulum where they would bind to Class I MHCmolecules. Eventually, the DCs would present the influenza antigen onthe surface in the Class I MHC molecule. Activated, influenzaantigen-loaded antigen presenting cells would migrate to lymphocytebearing secondary organs such as the regional lymph nodes or the spleen.During the two weeks of continuous release of the antigen/CD40 fusionprotein, CD8 cytotoxic T cell lymphocytes competent to recognize andkill cells, which carried the antigens, would be expanded in the lymphnodes and spleen by the presence of the activated and antigen loadeddendritic cells. The continuous nature of the stimulation and theexpansion of the influenza antigen specific cytotoxic T cells by thecontinuous release from the vector infected cells is believed togenerate an immune response which would be greater in magnitude than ispossible using a vector which carried an antigen/CD40 ligand which isnon-secretory.

Methods to test the novel influenza vaccines, proteins and vaccinationmethods of the present invention may include in vitro and in vivomethods well known in the art, method as described herein, and may alsoinvolve methods utilizing mouse models and viruses that have been mouseadapted. For example, the laboratories of M. K. Sim and Vincent Chow atthe National University of Singapore's School of Medicine have developeda mouse model for the study of the Ad-sig-H3N2/ecdCD40L vaccine againstH3N2 influenza A virus. The A/Aichi/2/68 (H3N2) strain (Arch. Virol.1983 75:17-27; Virology 1995 212:526-534) was mouse-adapted by passagingthrough several batches of 2 month old mice via intranasaladministration. Briefly, influenza virus was initially administeredintranasally to 6 week-old female BALB/c mice. Two days later, theanimals were sacrificed, lung homogenates prepared and labeled as“passage-1 lung homogenate”. A second batch of BALB/c mice was infectedwith 50 μL of passage-1 lung homogenate, and the process of passagingwas repeated. The virus virulence and titer in each passage of lunghomogenate was monitored by assaying its infectivity in Madin-Darbycanine kidney (MDCK) cells, host cells which are permissive forinfluenza A virus replication. The influenza A virus titer increasedprogressively with each passage. Thus it is possible to adapt an H3N2influenza virus to the Balb/C mouse strain.

In similar fashion, an H5N1 influenza virus may also be adapted to amouse strain by passaging through several batches of 2 month old micevia intranasal administration, using the method of Chow described above.Briefly, the virus is initially administered intranasally to 6 week-oldfemale BALB/c mice. Two days later, the animals are sacrificed, lunghomogenates prepared and labeled as “passage-1 lung homogenate,” Asecond batch of BALB/c mice is infected with 50 μL of passage-1 lunghomogenate, and the process of passaging is repeated. The virusvirulence and titer in each passage of lung homogenate is monitored byassaying its infectivity in host cells which are permissive for H5N1virus replication.

In a further aspect, the invention provides methods of effectivelyimmunizing older individuals by administering an expression vectorencoding a secretable fusion protein which includes an antigen and CD40ligand. Older individuals which may realize an improved immune responseusing the methods of the invention as compared to other immunizationmethods are at least 50 years of age, or even at least 55 years of ageor even yet at least 60 years of age.

The invention methods are advantageous over traditional vaccination inproviding increased immune responses in older individuals. The benefitsof immunizing older individuals, at least 50 years of age, or event atleast 55 years of age or even at least 60 years of age is afforded forantigens including cancer antigens (tumor associated or tumor specific)or infectious agent antigens. Infectious agent antigens may bebacterial, viral, fungal, protozoan, and the like. Viral antigens may bean influenza antigen as described above. The viral antigen may be from ahuman papilloma virus. The viral antigen may be the E6 or E7 protein ofhuman papilloma virus.

The term “tumor associated antigen” (TAA) as used herein refers to aprotein which is present on tumor cells, and on normal cells duringfetal life (onco-fetal antigen), after birth in selected organs, or onmany normal cells, but at much lower concentration than on tumor cells.A variety of TAA are described that can be used in the invention methodsfor immunizing older individuals are described in U.S. PatentApplication Publication US 2005-0226888 (application Ser. No.11/009,533) titled “Methods for Generating Immunity to Antigen,” filedDec. 10, 2004. The patent publication describes various mucin TAAincluding MUC1 and tandem repeats of the mucin VNTR region, and HER2(neu). Other TAA include but are not limited to carcinoembryonicantigen, prostate specific antigen, CA 125, CA 15-3, and EFGr.

As used herein, tumor specific antigen (TSA) (aka, “tumor-specifictransplantation antigen or TSTA) refers to a protein absent from normalcells. TSAs usually appear when an infecting virus has caused the cellto become immortal and to express a viral antigen(s). An exemplary viralTSA is the E6 or E7 proteins of HPV type 16. TSAs not induced by virusesinclude idiotypes the immunoglobulin idiotypes associated with B celllymphomas or the T cell receptor (TCR) on T cell lymphomas.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

EXAMPLES

1. Construction of Adenoviral Expression Vectors (GeneralConsiderations)

Details for constructing adenoviral expression vectors containing atranscription unit encoding a fusion protein that includes an antigenfused to CD40 ligand can be found in U.S. Patent Application PublicationUS 2005-0226888 (application Ser. No. 11/009,533) titled “Methods forGenerating Immunity to Antigen,” filed Dec. 10, 2004. This publicationdescribes the preparation of expression vector sig-ecdhMUC1-ΔCtΔTmCD40L,which encodes a signal sequence (from an Ig kappa chain) followed by theextracellular domain of human MUC1 which is connected via a linker to afragment of the CD40 ligand (human or mouse), which contains theextracellular domain without the transmembrane or cytoplasmic domains.The fusion protein was engineered to be secreted from vector infectedcells by the addition of the kappa chain signal sequence to theamino-terminal end of the fusion protein. As described therein, thetranscription unit was introduced into the E1 gene region of theadenoviral vector backbone. After the adenoviral vector particles weregenerated in HEK 293 cells, the vector DNA was purified by cesiumchloride gradient centrifugation. The presence of the signal peptide inthe adenoviral vector was confirmed by restriction enzyme analysis andby DNA sequencing.

U.S. Patent Application Publication US 2005-0226888 also describes howto prepare a transcription unit that includes DNA encoding the signalsequence of the mouse IgG kappa chain gene upstream of DNA encodinghuman MUC-1 (“sig-ecdhMUC-1”). The transcription unit is formed with theassistance of pShuttle-ΔCtΔTmCD40L (no signal sequence and murine CD40L)as described in U.S. Patent Application Publication US 2005-0226888.Primers are used to encode spacer (linker) between the antigen and CD40L(a 10 residue spacer LENDAQAPKS; single letter code; SEQ ID NO:48). Thesig-ecdhMUC 1/ΔCtΔTmCD40L encoding DNA was cut from the pCDNA3TOPOvector using HindIII-XbaI restriction and inserted into pShuttle-CMV(see Murphy et al., Prostate 38: 73-78, 1999) downstream of the CMVpromoter. The plasmid is designated pShuttle-sig-ecdhMUC1-ΔCtΔTmCD40Land contains transcription unit sig-ecdhMUC1-ΔCtΔTmCD40L which encodesthe mouse IgG kappa chain secretory signal followed by the extracellulardomain of human MUC1 followed by a 10 amino acid linker with(LENDAQAPKS; SEQ ID NO:48) followed by murine CD40 ligand residues52-260.

U.S. Patent Application Publication US 2005-0226888 also describescloning the mouse HSF1 trimer domain which was added between theecdhMUC1 (antigen) encoding DNA and ΔCtΔTm CD40L by PCR. Theconstruction design includes use of a spacer on each end of the trimersegment. U.S. Patent Application Publication US 2005-0226888 alsodescribes use of PCR to add a His tag encoding sequence added to the endof the ΔCtΔTm CD40L.

The recombinant adenoviral vectors were generated using the AdEasyvector system (Stratagene, San Diego, Calif.). Briefly the resultingplasmid pShuttle-sig-ecdhMUC1-ΔCtΔTmCD40L, and other control adenoviralvectors were linearized with Pme I and co-transformed into E. colistrain BJ5183 together with pAdEasy-1, the viral DNA plasmid.Recombinants were selected with kanamycin and screened by restrictionenzyme analysis. The recombinant adenoviral construct was then cleavedwith Pac I to expose its Inverted Terminal Repeats (ITR) and transfectedinto 293A cells to produce viral particles. The titer of recombinantadenovirus was determined by the Tissue culture Infectious Dose (TCID₅₀)method.

2. Production of the Fusion Protein and Vector

The influenza virus antigen fusion protein was produced directly from anadenoviral vector that carries the expression cassette of the fusiongene encoding the fusion protein. The production cells (e.g. 293 cellline) at 80% confluency in growth medium were infected with the viralvector at the ratio of 10-100 viral particles per cell. The infectedcells were further cultured for 48-72 hours, when the viral vectorspropagated in the cells and the tumor antigen fusion proteins wereexpressed in the cells and secreted into culture media. The infectedcells were collected when 70-90% of them showed cytopathic effect (CPE).The cell culture media was collected separately. Cell lysates wereprepared through 3-time freeze-and-thaw cycles. The viral particles wereisolated via the standard procedure (see e.g., PNAS 2003100:15101-15106; Blood 2004 104:2704-2713)). The tumor antigen fusionproteins were purified through affinity chromatograph from the collectedcell media.

The fusion protein also were produced in bacterial cells as follows. ThecDNA encoding the fusion protein was subcloned into the pTriEx-2 hygroVectors (Novagen). Competent cells (Rosetta™ cells, Novagen Inc.) weretransformed with the resulting plasmid. Following incubation of thecells in IPTG supplemented medium for 4 hours, a cell lysate wasprepared using the CelLytic™ B Plus Kit (Sigma). The fusion protein waspurified from the soluble fraction by HIS-select Nickel Affinity Gel(Sigma). Then, the protein was concentrated and desalted bycentrifugation through an Ultrafree-15 Biomax-50 filter (Millipore) andeluted with PBS.

3. Construction of Adenoviral Vectors Encoding HPV E7-CD40 Ligand FusionProtein.

E7 is a protein encoded by the human papilloma virus which appears onall HPV associated dysplastic and neoplastic cells. The transcriptionunit included DNA encoding the signal peptide from the HGH gene,upstream of DNA encoding the full-length HPV type 16 E7 protein,consisting of 98 amino acids and having the following amino acidsequence:

(SEQ ID NO: 49) MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP.

The coding sequence for this E7 protein was upstream of the codingsequence of a 10 aa spacer, which was upstream of the coding sequence ofthe coding sequence of ΔCtΔTmCD44L in the transcription unit.

Construction of an adenoviral vector expressing a transcription unitfusion protein constituting E7 linked to a secretable form of CD40ligand has been described See, e.g., U.S. Patent Application PublicationUS 2005-0226888 (application Ser. No. 11/009,533) titled “Methods forGenerating Immunity to Antigen,” filed Dec. 10, 2004. This approach isdetailed below.

a) Construction of pShuttle-sp-ΔCtΔTmCD40L(No Signal Sequence).

Plasmid pDC406-mCD40L was purchased from the American Type CultureCollection. A pair of PCR primers (SEQ ID NOs:50 and 51) was designed toamplify the mouse CD40 ligand from position 52 to 260 (i.e., without thecytoplasmic and transmembrane domains) and include sequence encoding alinker (indicated as “+ spacer”) at the 5′ end of the amplicon.

Mouse ΔCtΔTmCD40L+ spacer forward primer (MCD40LSPF) (Xho I recognitionsite in bold and underlined; spacer sequence underlined (includes theXho I site); CD40L sequence italicized):

(SEQ ID NO: 50) 5′-CCG CTCGAG AAC GAC GCA CAA GCA CCA AAA TCA AAG GTCGAA GAG GAA GTA AAC-3′.Mouse CD40L reverse primer (MCD40LR) (Xba I recognition site in bold andunderlined)

(SEQ ID NO: 51) 5′-CCC TCTAGA  ATCAGAGTTTCACTAAGCCAA-3′.

The forward primer MCD40LSPF encoded a 10 residue spacer (LENDAQAPKS;single letter code; SEQ ID NO:48) to be located between the tumorantigen and the CD40 ligand (mCD40L) of the transcription unit. PCR wasperformed using the forward and reverse primers (SEQ ID NOs:50 and 51)and plasmid pDC406-mCD40L as the template under the followingconditions: hold 3 min at 94° C.; cycle 94° C. for 45 sec, 55° C. for 45sec, 72° C. for 70 sec (30 cycles); hold 7 min at 72° C.; and hold at 4°C. This PCR resulted in a fragment “spacer+ΔCtΔTmCD40L,” which wasinserted into the plasmid pShuttle-CM V (Murphy et al. Prostate 38:73-8,1999) after restriction endonuclease digestion with Xba I (TCTAGA) andXho I (CTCGAG). This vector is designated pShuttle-sp-ΔCtΔTmCD40L(nosignal sequence).

A vector was produced that was otherwise the same except that it encodedfull length CD40L rather than the truncated form. This vector was madeusing a CD40 forward primer that annealed to the starting codons ofmurine CD40L. This vector is designated pShuttle-mCD40L (no signalsequence).

b) Construction of pShuttle-E7-sp-ΔCtΔTmCD40L(no signal sequence).

pShuttle-E7-ΔCtΔTmCD40L (no signal sequence) was prepared by insertingHPV-16 E7 upstream of the CD40 ligand sequence as follows: Sequenceencoding the full-length HPV-16 E7 protein was obtained by PCRamplifying from the HPV viral genome using the following primers:

HPV 16 E 7 forward primer (SEQ ID NO: 52) 5′-ATTT GCGGCCGC TGTAATCATGCATGGAGA-3′; HPV E7 reverse primer (SEQ ID NO: 53) 5-CCCTCGAG  TTATGGTTTCTGAGAACAGAT-3′.

PCR was performed using the above primers and the HPV 16 viral genome astemplate under the following conditions: hold 3 min at 94° C.; cycle 94°C. for 40 sec, 58° C. for 40 sec, 72° C. for 40 sec (30 cycles); hold 7min at 72° C.; and hold at 4° C. The resulting amplicon was HPV 16 E7encoding DNA with Not I and Xho I restriction sites at the 5′ and 3′ends, respectively. The E7 DNA was inserted into thepShuttle-sp-ΔCtΔTmCD40L(no signal sequence) vector between the CMVpromoter and directly 5′ to the spacer of the spacer-ΔCtΔTmCD401,sequence using the Not I (GCGGCCGC) and Xho I (CTCGAG) restrictionsites. The resulting plasmid is designated pShuttle-E7-ΔCtΔTmCD40L(nosignal sequence).

c) Construction of pShuttle-HGH/E7-sp-ΔCtΔTmCD40L.

The pShuttle-E7-sp-ΔCtΔTmCD40L(no signal sequence) vector was used forinsertion of the HGH signal sequence, upstream of E7 to generateHGHIE7-sp-ΔCtΔTmCD40L, described as follows.

DNA encoding the human growth hormone signal sequenceMATGSRTSLLLAFGLECLPWLQEGSA (single letter amino acid code) (SEQ IDNO:54) was prepared by annealing phosphorylated oligonucleotides (SEQ IDNOs:55 and 56) to generate the full 26 amino acid HGH sequence with BglII and Not1 overhangs.

Growth hormone signal upper strand (coding sequence in italics

(SEQ ID NO: 55) 5′-GATCT CCACC ATG GCT ACA GGC TCC CGG ACG TCC CTG CTCCTG GCT TTT GGC CTG CTC TGC CTG CCC TGG CTT CAA GAG GGC AGT GCC GGC -3′.Growth hormone signal lower strand:

(SEQ ID NO: 56) 3′-A GGTGG TAC CGA TGT CCG AGG GCC TGC AGG GAC GAG GACCGA AAA CCG GAC GAG ACG GAC GGG ACC GAA GTT CTC CCG TCA CGG CCGCCGG-5′.

Synthetic HGH signal sequence was prepared by annealing the above upperand lower strand oligos. The oligos were dissolved in 50 μl H₂O (about 3mg/ml). 1 μl from each oligo (upper and lower strand) was added to 48 μlannealing buffer (100 mM potassium acetate. 30 mM HEPES-KOH pH 7.4, and2 mM Mg-acetate) incubated for 4 minutes at 95° C., 10 minutes at 70° C.and slowly cooled to about 4° C. The annealed DNA was phosphorylatedusing T4 PNK (polynucleotide kinase) under standard conditions.

The HGH signal sequence with Bgl II and Not I overhangs was inserted viaBgl II and Not I into pShuttle-E7-sp-66 CtΔTmCD40L(no signal sequence)to yield pShuttle-HGH/E7-sp-ΔCtΔTmCD40L. Thus, the transcription unitHGH/E7-sp-ΔCtΔTmCD40L encodes the HGH secretory signal followed by thefill length HPV type 16 E7 followed by a 10 amino acid linker with(LENDAQAPKS; SEQ ID NO:48) followed by murine CD40 ligand residues52-260.

d) Construction of pShuttle-K/E7-sp-ΔCtΔTmCD40L

A transcription unit that included DNA encoding the signal sequence ofthe mouse IgG kappa chain gene upstream of DNA encoding the fill lengthHPV type 16 E7 protein (“K/E7”) was generated by PCR using HPV16 plasmidand the following primers:

(primer 1) (SEQ ID NO: 57) 5′-ACG ATG GAG ACA GAC ACA CTC CTG CTA TGGGTA CTG CTG-3′; (primer 2) (SEQ ID NO: 58) 5′-TC CTG CTA TGG GTA CTG CTGCTC TGG GTT CCA GGT TC-3′; (primer 3) (SEQ ID NO: 59) 5′-TG CTC TGG GTTCCA GGT TCC ACT GGT GAC ATG CAT G-3′; (primer 4) (SEQ ID NO: 60) 5′-TGGGTT CCA GGT TCC ACT GGT GAC ATG CAT GGA G AT ACA CCT AC-3′; and (primer5) (SEQ ID NO: 61) 5′-CCG CTC GAG  TGG TTT CTG AGA ACA GAT GGG GCAC-3.′.K/E7 with the upstream kappa signal sequence was generated by fourrounds of PCR amplification (1^(st) round: primers 4+5; 2^(nd) round:add primer 3; 3^(rd) round: add primer 2; 4^(th) round: add primer 1).The K/E7 encoding DNA was cloned into the pcDNA™ 3.1 TOPO vector(Invitrogen, San Diego, Calif.) forming pcDNA-K/E7.

A DNA fragment that contained coding sequence for the 10 aa spacerupstream of mouse CD40 ligand from which the transmembrane andcytoplasmic domain had been deleted (-sp-ΔCtΔTmCD40L) was generated froma mouse CD40 ligand cDNA plasmid, pDC406-mCD40L (American Type CultureCollection), using the following PCR primers:

(SEQ ID NO: 62) 5′-CCG CTCGAG  AAC GAC GCA CAA GCA CCA AAA AGC AAG GTCGAA GAG GAA GTA AAC CTT C-3′; and (SEQ ID NO: 63) 5′-CGCGCCGCGCGCTAG

 GAGTTTGAGTAAGCCAAAAGATGAG-3′ (high fidelity PCR kit, Roche).

Fragment sp-ΔCtΔTmCD40L was digested with Xba I and XhoI restrictionendonucleases and then ligated into pcDNA-KIE7. The K/E7-sp-ΔCtΔTmCD40Lfragment was cut from the pcDNA vector and inserted into the pShuttleplasmid using Hind III and Xba I sites (pShuttle K/E7-sp-ΔCtΔTmCD40L).Thus, the K/E7-sp-ΔCtΔTmCD40L fragment includes the kappa chainsecretory signal followed by the full length HPV type 16 E7 followed bya 10 amino acid linker (LENDAQAPKS; SEQ ID NO:48) followed by murineCD40 ligand residues 52-260.

e) Construction of pShuttle-HGH/E7-CD40L.

Adenoviral vector encoding a fusion protein with E7 upstream of fulllength mouse CD40L (with no intervening linker) was made using primersto amplify full length mouse CD40L using PCR. The following primers wereused:

forward primer: (SEQ ID NO: 64) 5′-GAGAC CTCGAG  CAGTCA GC ATGATAGAAACATACAGC CAACCTTCCC-3′; reverse primer: (SEQ ID NO: 65)5′-CGCGCCGCGCGC CCC TCTAGA  TCA GAG TTT GAG TAA GCC AAA AGA TGA G-3′.

Amplified DNA was initially subcloned into the pcDNA3-K/E7 vector withXba I and XhoI restriction endonucleases. The full length CD40L gene orΔCtΔTmCD40L was directionally cloned into the pShuttle plasmid with theHind III and Xba I sites.

f) Construction of pShuttle-HGH/E7-sp-ΔCtΔTmCD40L(human).

A vector encoding an E7/human CD40 ligand fusion protein(pShuttle-HGH/E7-sp-ΔCtΔTmCD40L(human)) is described as follows. Primersfor amplifying human ΔCtΔTMCD40L+ spacer using a human CD40 ligand cDNAtemplate are set forth below.

Human ΔCtΔTmCD40L+ spacer forward primer (HCD40LSPF) (CD40L sequenceitalicized): (SEQ ID NO: 66)

CAT AGA AGG TTG GAC-3′; Human CD40L reverse primer (HCD40LR)(SEQ ID NO: 67) 5′-CCC  TCTAGA  TCAGAGTTTGAGTAAGCCAAAGGAC-3′.PCR is performed using the above primers and the plasmid pDC406-hCD40Las template under the following conditions: hold 3 min at 94° C.; cycle94° C. for 45 sec, 52° C. for 45 sec, 72° C. for 70 sec (30 cycles);hold 7 min at 72° C.; and hold at 4° C. This amplification results inthe “-sp-ΔCtΔTmCD40L(human)” fragment, which encodes 44-261 of humanCD40L and an amino terminal 10 aa spacer. The forward primer HCD40LSPFencodes a 10 residue spacer (LENDAQAPKS; single letter code; SEQ IDNO:48) to be located between the tumor antigen and the CD40 ligand(hCD40L) of the transcription unit. The “sp-ΔCtΔTmCD40L(human)” fragmentis then inserted into the plasmid pShuttle-CMV (Murphy G P, el atProstate 38: 73-78 (1999)) after restriction endonuclease digestion withXba I (AAGCTT) and Xho I (CTCGAG). This vector is designatedpShuttle-sp-ΔCtΔTmCD40L(human)(no signal sequence). Modification ofpShuttle-sp-ΔCtΔTmCD40L(human)(no signal sequence) to include the HPV-16E7 upstream of the human CD40 ligand sequence is accomplishedessentially as described above for the murine CD40 ligand encodingvectors. The resulting plasmid is designatedpShuttle-E7-sp-ΔCtΔTmCD40L(human)(no signal sequence) and is used forinsertion of the HGH signal sequence upstream of E7 to generateHGH/E7-sp-ΔCtΔTmCD40L(human). Thus, the transcription unitHGH/E7-sp-ΔCtΔTmCD40L(human) encodes the HGH secretory signal, followedby the full length HPV type 16 E7, followed by a 10 amino acid linker(LENDAQAPKS; SEQ ID NO:48) followed by human CD40 ligand representingresidues 44-261.

h) Recombinant Adenovirus

The recombinant adenoviral vectors were generated using the AdEasyvector system (Strategene, San Diego, Calif.). Briefly, the resultingplasmids pShuttle-HGH/E7-sp-ΔCtΔTmCD40L, pShuttle-FIGH/CD40L,pShuttle-HGH/E7-CD40L, and pShuttle-HGH/E7 were linearized by Pme Idigestion and then co-transformed into E. coli strain BJ5183 togetherwith pAdEasy-1. Recombinants were selected with kanamycin and screenedby restriction enzyme analysis. The recombinant adenoviral construct wasthen cleaved with Pac I, and transfected into 293A cells to produceviral particles. The titer of recombinant adenovirus was decided by thetissue culture infectious dose 50 (TCID₅₀) method.

4. Purification of Recombinant E7/ecdCD40L Protein in Bacterial Systemand E7 Assay Methods

a) Purification of Recombinant E7/ecdCD40L Protein

The E7/ecdCD40L fusion cDNAs were inserted into the expression vectorpTri by XcmI and NotI sites. The expression bacterial cell line Rosetta(DE3) was transfected by pTri E7/ecdCD40L vectors and induced by IPTGfor 3 hours at 37° C. The bacterial pallets were harvested and purifiedby His Selected Nickel Affinity Gel (Sigma).

b) Flow Cytometry Analysis of T Regulatory Cells

To quantify T regulatory cells, the CD4 T cells from lymph node, spleenor tumor nodule were respectively stained by two different kinds ofmarkers, CD4CD25 and CD4FOXP3, with FITC- or PE-conjugated anti-mousemonoclonal antibodies (Pharmingen, eBiscience) for 30 min on ice, priorto immunostaining with labeled antibodies. The T cells were firstincubated with a Fc-γ blocking antibody (anti-mouse CD16/CD32 antibody)to avoid the nonspecific binding of mAbs to Fc-γ receptors. The cellswere then washed twice, fixed in 4% paraformaldehyde, and analyzed usinga Becton Dickinson flow cytometer (FACS Calipur).

c) Tetramer Staining

PE-labeled H-2Db tetramer containing HPV16 E7 49-57 peptide (RAHYNIVIT)(SEQ ID NO: 68) was purchased from Beckman Coulter and used for theanalysis of peptide specific CTL immunity. Ten days after immunization,1×106 erythrocyte-depleted spleen cells were stained by 10 ul oftetramer together with 1/100 diluted fluorescein isothiocyanate(FITC)-anti mouse CD8a (clone53-6.7, BD Pharmingen) in 100 ul PBSsupplemented with 3% FCS, incubated at room temperature for 30 minutes,and then washed with 3 ml of PBS. Following centrifugation, the cellpellet was resuspended in 500 ul of PBS/0.5% paraformaldehyde for FACSanalysis. Tetramer positive and CD8+ cells are shown as a percentage oftotal spleen cells.

d) Cytokine Profile by ELISPOT Assays

The presence of E7-specific effector T cells in the immunized mice wasalso assessed by carrying out ELISPOT assays, as described previously.Briefly, splenocytes obtained from mice vaccinated with each of thedifferent vectors were-restimulated in vitro by culture with the TC-1cell line (responder-to-stimulator ratio=25/1) in the presence of 10U/ml IL-2 for 48 hours. Re-stimulated splenocytes were then plated in96-well nitrocellulose filter plates (5×10⁴ cells in 100 microliters).The wells were pre-coated with rat anti-mouse anti-IFN-antibody oranti-IL-4 antibody. After incubation for 24 hours at 37° C./5% CO₂, theplates were then washed with PBS, and the presence of cytokine-producingspleen cells was detected by incubation at 4° C. with biotinylated goatanti-rat secondary antibody, followed by 100 microliter/well ofhorseradish peroxidase avidin D. To this was added 150 microliter/wellfreshly prepared substrate buffer (0.4 mg/ml 3-amino-9-ethyl-carbazolein a total of 50 ml 0.05 mol/In sodium acetate buffer) and 20 microliter30% H₂O₂. The stained spots corresponding to IFN producing cells or to1L-4 producing cells, were enumerated under a dissecting microscope.

e) Cytotoxicity Assay

Mononuclear cells from the spleens of these mice were incubated withmitomycin C-treated TC-1 cells in RPMI 1640 medium, supplemented with10% fetal bovine serum (FBS), 5 mM 2-mercaptoethanol, 2 mM glutamine, 1mM pyruvate, and nonessential amino acids for 5 days. To perform thecytotoxicity assay, firstly TC-1 tumor cells/(target cells) were labeledwith the red-fluorescent dye PKH26 (Sigma, St Louis, Mo.) according tothe manufacturer's specifications. In brief, the target cells werewashed in PBS then resuspended at 10⁷ cells/mi in solution. PKH26 dyewas added to a final concentration of 2 μM, mixed and incubated at roomtemperature. After 5 min, the reaction was quenched with 3 volumes ofFCS and the cells were washed an additional 3 times in RPMI/10%FCSmedium then 5×10³ labeled TC-1 cells were incubated with the stimulatedsplenic mononuclear cells (effector cells) at a differenteffector/target ratio for 4 hours at 37° C., in culture media containing5% FBS. At the end of the incubation, mononuclear cell-mediatedcytotoxicity was stained for intracellular Caspase-3 according to themanufacturer's protocol(BD PharMingen), double positive cells wasdetermined by flow cytometry on live gated PKE26⁺ cells.

f) In Vivo Efficacy Experiment in Mouse Model

Mice (5 or 10 per group) were challenged by subcutaneous injection of5×10⁵ TC-1 cells were injected subcutaneously. On the next day, the micewere vaccinated via SC injection with 1×10⁸ PFU Ad-sig-E7/ecdCD40L. Oneweek later, mice were boosted with the same adenoviral vector regimen asthe first vaccination or followed by SC injections of the 10 ugrecombinant E7/ecdCD40L protein every week. Tumor volumes were measuredin centimeters by caliper. One month later, the tumor free mice wererechallenged by 1×10⁷ TC-1 cells and the tumor volume was calculated astumor volume=length×(width²)/2 (this assumes an ellipitical shape).

g) Statistics

All parameters were analyzed using Student's t test, or ANOVA followedby Scheffé's procedure for multiple comparisons as post-hoc analysis;all data shown is presented as mean ±S.E. of the mean (S.E.).

5. Inducing Immunity Against a Viral Antigens in Young (Two Month Old)Mice with Ad-sig-E7JecdCD40L Vector.

Two sc injections (seven days apart), each 10 μg/mouse of theAd-sig-E7/ecdCD40L adenoviral vector were given to six week old C57BLI6Jmice which were evaluated for induction of resistance to engraftment ofthe E7 positive TC-1 cells (see PNAS 2003 100:15101-15106; Blood 2004104:2704-2713). Injected mice were challenged with 500,000 E7 positiveTC-1 cells and 500,000 E7 negative EL-4 cells implanted subcutaneously(at a different site than the adenoviral vector injection site) sevendays following the last vaccination injection. The growth of E7 negativeEL-4 cells (measured by tumor volume) was not suppressed in theAd-sig-E7lecdCD40L injected mice, while E7-positive syngeneic TC-1 cellgrowth was completely suppressed in injected mice. FIG. 6. These resultsindicate that the Ad-sig-E7/ecdCD40L vector induces a specific immuneresponse directed to the E7 viral antigen in young 2 month old animals.

6. Ad-sig-E7/ecdCD40L Vector Induced Immunity in Young (Two Month Old)Mice is CD8 Dependent

To test if the Ad-sig-E7/ecdCD40L vector injections in 2 month old miceinduced immunological memory cells, splenic T lymphocytes were collectedfrom C57BL/6J mice which had survived for a full year after vaccinationwith the Ad-sig-E7/ecdCD40L vector and challenge with the E7 positiveTC-1 cells. The test C57BL/6 nude mice (n=7) were injected sc with500,000 TC-1 cells and then injected intraperitoneally five days laterwith 10×10⁶ splenic lymphocytes from mice which had been vaccinated withthe Ad-sig-E7/ecdCD40L vector one year earlier. As shown in FIG. 7, onlytransient growth of the TC-1 cells (solid diamonds) occurred in theC57BL/6 nu/nu mice which had received intraperitoneal injections of theT cells from the Ad-sig-E7/ecdCD40L vaccinated mice (♦). In contrast,the TC-1 cells grew well (line defined by the solid squares (▪)) incontrol C57BL/6 nu/nu mice which were injected intraperitoneally with10×10⁶ splenic T cells from unsensitized donor C57BL/6 mice five daysafter injection of TC-1 cells. These results show that the immunityinduced by the vector injections involves memory cells.

To test whether this immune resistance was dependent on CD8+ or CD4+ Tcell lymphocytes, donor immunocompetent C57BL/6 mice were injected scwith the Ad-sig-E7/ecdCD40L vector at days zero and seven. Seven dayslater (day 14), the mice were injected se with 100,000 E7 positive TC-1cells. As shown below in FIG. 8, the C57BL/6 donor mice were treated invivo with antibodies which were cytolytic and specific for CD4 (soliddiamonds) or CD8 (solid triangles) positive cells to deplete therespective T cell population 5, 3, and 1 days prior to the vectorinjection, and every six days after the sc injection of theAd-sig-E7/ecdCD40L vector, and also on days 6, 7, 8, 10, 12, and 14 daysfollowing the injection of the TC-1 cells. Then the sensitized CD8+ Tcells from the CD4 depleted vaccinated mice (see solid diamonds in FIG.8) or CD4+ T cell lymphocytes from the CD8 depleted vaccinated mice (seesolid triangles in FIG. 8) from sensitized C57BL/6 donors were injectedintraperitoneally into C57BL nude mice 7 days after subcutaneousinjection of 1×10⁵ TC-1 cells. A third group of C57BL/6 nude mice, whichwere control mice, did not receive passive transfer of T cells (seesolid squares in FIG. 8) 7 days following subcutaneous injection of TC-1cells. The C57BL/6 nu/nu mice injected with CD8+T cells from theAd-sig-E7/ecdCD40L injected donor animals survived statisticallysignificantly longer than did the other groups, Thus, the induction ofimmunity to TAA positive tumor cells by the injection of theAd-sig-TAA/ecdCD40L vector was dependent on CD8+ T cell lymphocytes andnot dependent on CD4 cells. This vaccine is therefore useful forcircumventing the CD4 defects in old people for influenza vaccines.

7. Ad-sig-E7/ecdCD40L Vector Increases E7 Viral Antigen SpecificCytotoxic T Lymphocytes in Young (Two Month Old) Mice

Spleen cells were isolated from C57Bl/6 mice (from Harlan) before and 7days after two injections with 1×10⁸PFU of the Ad-sig-E7lecdCD40Lvector. The level of the E7-specific T cells was determined by tetramerassay as described above.

T cells increased three fold in the spleen following theAd-sig-E7/ecdCD40L vector injection, whereas no significant increasesoccurred with the other control vectors (Ad-E7/wtCD40L; ad-wtCD40L,Ad-sig-ecdCD40L) including the Ad-E7/wtCD40L, in which the E7/CD40Lprotein was not secretable from the infected cells. FIG. 9.

8. Ad-sig-E7/ecdCD40L Vector Induces Viral Antigen Immunity in Old (18Month Old) Mice

The ability to elicit viral antigen immunity in old (18 month old) miceusing The Ad-sig-E7/ecdCD40L vector was evaluated. TheAd-sig-E7/ecdCD40L vector was injected once sc with 1×10⁸ IU. At days 7,14 and 21 after the vector injection, an sc injection of the E7/ecdCD40Lprotein (10 micrograms/injection) was given as a booster. Seven dayslater, mice were sacrificed and the level of E7 specific T cells in thespleen determined by ELISPOT assay.

The levels of interferon-gamma positive cells/100,000 splenocytes wasincreased to 225 in the old mice and 725 in young mice, while IL-4 cellnumbers increased to over 100 antigen specific T cells/100,000splenocytes. FIG. 10 Although the magnitude of the induction of antigenspecific T cells in the 18 month old mice was less than that seen in the2 month old mice, the absolute magnitude of the response in the 18 monthold mice is in the range induced by most other vaccines in young miceand is clearly sufficient to produce'a robust immune response.

The increase in the percentage of antigen specific T cells to total CD8T cells in the tumor tissue in old mice was measured via E7 tetramersbefore and after vaccination. The Ad-sig-E7/ecdCD40L vaccine induced thelevel of antigen specific T cells in the tumor tissue by 10 fold (FIG.11).

The increase in the number of T cells as a percentage of the totalnumber of cells in the tumor tissue following vaccination in the oldmice was also measured. The increase of the percentage of T cellsincreased over 10 fold after the vaccination in the old mice (FIG. 12).

The level of increase of antigen specific cytotoxic T lymphocytes(“CTLs”) induced by vaccination in 2 month and 18 month old mice wasalso evaluated using the protein boost immunization scheme. Increases inantigen specific CTLs following vaccination in the old as well as theyoung animals is shown in FIG. 13. Again, the level of the increase ofthe CTLs seen in the 18 month old mice was less than that seen in the 2month old mice, but the absolute magnitude of the induction wasimpressive in the 18 month old mice.

9. Immune Response Induced by the V, VP, VPP, and VPPP VaccinationRegimens with the Ad-sig-E7/ecdC40L Vector Prime in 2 Month Old(“Young”) Mice.

Two month old C57BL6 mice were vaccinated using one of the followingfour vaccination regimens: a single subcutaneous injection ofAd-sig-E7/ecdCD40L (“V”), or a single subcutaneous vector injectionfollowed by one boost with fusion protein (“VP”), two boosts with fusionprotein (“VPP”), or three boosts with fusion protein (“VPPP”), in whicheach boost is a weekly subcutaneous injections of the E7/ecdCD40L fusionprotein. The levels of E7-specific splenic CD8 T cells and E7-specificantibody levels in serum were determined. As shown in FIG. 27, thelevels of the E7-specific splenic CD8 T cells of the vaccinated miceincreased going from V to VP to VPP and to VPPP. VP, VPP and VPPP aresignificantly different from each other (p<0.05). As shown in FIG. 28,the levels of the E7 specific serum antibodies against the E7 B cellepitope ElDGPAGQAEPDRAHYNIVTFCCI(CD of the vaccinated mice increasedsignificantly with the number of fusion protein boosts after the initialvector injection (VP<VPP<VPPP). At a dilution factor of 1/1000, thedifference of the serum antibody levels in the vaccinated versus theunvaccinated control group was statistically significant for VP mice(p=0.004); VPP mice(p<0.001); and VPPP mice(p<0.0001).

10. Immune Response Induced by the V, VP, VPP, and VPPP VaccinationRegimens with the Ad-sig-E7/ecdC40L Vector Prime in 18 Month Old (“Old”)Mice.

18 month old C57BL6 mice were vaccinated using one of the following fourvaccination regimens: a single subcutaneous injection ofAd-sig-E7/ecdCD40L (V), or a single subcutaneous vector injectionfollowed by one (VP), two (VPP) or three (VPPP) weekly subcutaneousinjections of the E7/ecdCD40L protein boost. The levels of E7-specificsplenic CD8 T cells and E7-specific antibody levels in serum weredetermined. As shown in FIG. 29, increases in the levels of theE7-specific splenic CDR T cells of the vaccinated mice were detectableonly at the VPP and VPPP level. As shown in FIG. 30, the levels of theE7-specific serum antibodies against the E7 B cell epitopeEIDGPAGQAEPDRAHYNIVTFCCKCD of the vaccinated mice increasedsignificantly only with three protein boost injections after the initialvector injection (VPPP).

11. Effect of the Ad-sig-TAA/ecdCD401, Vector Vaccine and Protein BoostAgainst Viral Antigen in Old (18 Month Old) Mice on Growth of CellsPositive for Viral Antigen

The suppression of E7 positive tumor growth in 18 month old mice wasalmost equal to the level of suppression of the tumor growth in 2 monthold mice with the vector vaccination alone. FIG. 14. The effect of theprotein boosts on the induction of the immune response induced by theAd-sig-E7/ecdCD40L vector was tested. Test C57B1/6J mice were injectedfirst with 1×10⁸ PFU Ad-sig-E7/ecdCD40I, vector followed by 3 proteinboosts (10 μg protein per injection) given at 7, 14 and 21 days afterthe vector injection.

The endpoint of these studies was in vivo suppression of the E7 tumorgrowth in C57Bl/6J mice, as measured by the percentage of mice whichremained tumor free. As shown in FIG. 15, the sc injection of theE7/ecdCD40L protein induced regressions of existing tumor and convertedtumor positive mice to tumor negative mice. These data suggested thatAd-sig-TAA/ecdCD40L by se vector and protein boost could induce completeregressions in existing tumor which was progressive in 18 month oldmice.

12. Effect of the Ad-sig-TAA/ecdCD40L Vector Vaccination in Old Mice onthe Levels of CD4 FOXP3 Negative Regulatory T Cells in Tumor Tissue

Increases CD4 FOXP3 negative regulatory T cells have been reported tolimit the degree to which vaccines suppress the degree of immuneresponse to vaccination. Decreases in the level of FOXP3 negativeregulatory CD4 T cells have been reported with vaccination. Thereforethe level of FOXP3 CD4 T cells in the tumor tissue before and after1×10⁸ PFU Ad-sig-E7/ecdCD40L vector (including the protein boosting) wasmeasured by FACS analysis. As shown in FIG. 16, vaccination in oldanimals decreased the CD4 FOXP3 negative regulatory T cells in the tumortissue by 2 fold.

13. Levels of CD8 Effector and CD4 Negative Regulatory T Cells in TumorTissue Following Ad-sig-rH2N/ecdCD40L Vector Vaccination in Young (2Month Old) Mice

The levels of antigen specific CD8 effector T cells in tumor tissue wasdetermined after vaccination with the Ad-sig-rH2N/ecdCD40L vector, thepreparation of which is described in U.S. Patent Application PublicationUS 2005-0226888 (application Ser. No. 11/009,533) titled “Methods forGenerating Immunity to Antigen,” filed Dec. 10, 2004. Subcutaneous tumornodules of rH2N.Tg mice were minced before and after two sc injectionsof the Ad-sig-rH2N/ecdCD40L vector. 1×10⁸PFU vector were administeredper injection; injections were given 7 days apart, and tumor noduleswere isolated 10 days after the last injection. Single cell suspensionswere generated from the tumor tissue after mincing, treated with 0.03%DNAse and then 0.14% collagenase I, and filtered through Nylon mesh. Thenumber of effector T cells isolated from the tumor tissue aftervaccination (CD8+, CD44+, LY6C+ and CD62L−) was increased roughly 5-7fold. FIG. 17. This data suggests that the suppression of the growth ofthe rH2N positive tumor cells in the rH2N.Tg mice followingAd-sig-rH2N/ecdCD40L vaccination is mediated in part by the traffickingof rH2N specific CD8 effector T cells into the tumor tissue.

RNA was isolated from the tumor infiltrating CD8 effector T cells andthe pattern of gene expression was compared before and after vaccinationusing the Affymetrix gene expression system. The expression level of the21 known chemokine receptors and ligands in the effector T cells whichwere infiltrating the tumor tissue was also examined. The levels of theCCL3 (2.8 fold increase) and the CCR5 (16 fold increase), which areinvolved in the targeting of I cells to the extravascular sites oftissue inflammation, were increased in the rH2N specific CD8 effector Tcells in the tumor tissue. The chemokine pathway plays a major role inthe trafficking of effector and memory T cells from the lymph nodesdraining sites of vaccination or infection to the tissue sites harboringinflammation or infection (Current Opinion 2003 15:343-348; CellAdhesion and Communication 1998 6:105-110).

14. Induction of Immunity with the Ad-sig-H5N1HA/ecdCD40L Vector Againstthe Hemagglutinin Protein of the H5N1 Avian Influenza Virus in 2 MonthOld Mice and 18 Month Old Mice

A portion of the HA sequence from an H5N1 influenza virus was used as anantigen in the Ad-sig-TAA-ecdCD40L vector, creating an Ad-sig-H5HA-ecdCD40L expression vector. As described below, this vector inducedan immune response in mice; a significant increase in the level of H5 HAspecific CD8 T cells was detected.

a) Construction of the Ad-sig-H5HA-ecdCD40L Expression Vector i)H5HA-ecdCD40L(mouse)

DNA (SEQ ID NO: 69) encoding a portion of a receptor binding regioncorresponding to amino acid residues 135-175 connected to residues230-250 (see FIG. 2, underlined) of the hemagglutinin (HA) protein ofthe H5N1 avian influenza strain (H5HA), first isolated in 1997 in HongKong from a child with a fatal respiratory illness (see, e.g., Science1998 279:393-96; J. Virology 1998 73: 2094-98; Emerging InfectiousDisease 2002.8(8):1-12) is shown below.

(SEQ ID NO: 69) 5′AAAAGTTCTTGGTCCAATCATGATGCCTCATCAGGGGTGAGCTCAGCATGTCCATACCTTGGGAGGTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTGCATACCCAACAGCTACTAGACCCAAAGTAAACGGGCAAAGTGGAAGAATGGAGTTCTTCTGGACAATTTTAAAG-3′.The above HA construct was generated using the following two primers:

forward primer: (SEQ ID NO: 70)5′AAAAGTTCTTGGTCCAATCATGATGCCTCATCAGGGGTGAGCTCAGCATGTCCATACCTTGGGAGGTCCTCCTTTTTCAGAAATGTGGTATGGCTT ATCAAAAAGAACAGTGC-3′and reverse primer: (SEQ ID NO: 71)5′CTTTAAAATTGTCCAGAAGAACTCCATTCTTCCACTTTGCCCGTTTACTTTGGGTCTAGTAGCTGTTGGGTATGCACTGTTCTTTTTGATAAGCCAT ACCAC-3′.

Double-stranded nucleic acid encoding e HA region was generated asfollows. The oligos were dissolved in 50 μl H₂O (about 3 mg/ml), 1 μlfrom each oligo (forward and reverse primers) was added to 48 μlannealing buffer plus 7 μL ddH₂O (100 m M Tris-HCl , 1M NaCl, 10 mMEDTA) incubated for 4 minutes at 95° C., 10 minutes at 70° C. and slowlycooled to about 4° C.

A transcription unit that included DNA encoding the signal sequence ofthe mouse IgG kappa chain gene upstream of DNA encoding the Avian HAantigen epitope was generated by PCR using the double-stranded HAgenerated in the previous paragraph and the following primers:

(SEQ ID NO: 72) 5′-CCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG GTA CTGCTG-3′; (SEQ ID NO: 73) 5′-TC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCAGGT TC-3′; (SEQ ID NO: 74) 5′-TG CTC TGG GTT CCA GGT TCC ACT GGT GAC ATGCAT G-3′; (SEQ ID NO: 75) 5′-TGG GTT CCA GGT TCC ACT GGT GAC ATGAAAAGTTCTTGGTCCAATCATGATGC-3′; and (SEQ ID NO: 76) 5′-CCGCTCGAG GCTTTAAAATTGTCCAGAAGAACTCC-3′.

K/Avian FluHA (i.e., kappa signal-H5 fragment) was generated by fourrounds of PCR amplification (1^(st) round: primers 4 +5; 2^(nd) round:add primer 3; 3^(rd) round: add primer 2; 4^(th) round: add primer 1),under the following conditions: hold 3 min at 94° C.; cycle 94° C. for30 sec, 55° C. for 30 sec, 72° C. for 30 sec (30 cycles); hold 7 min at72° C.; and hold at 4° C. The KlAvian FIuHA encoding DNA was cloned intothe pcDNA™ 3.1 TOPO vector (Invitrogen, San Diego, Calif.) formingpcDNA-K/Avian FluHA.

Primers for amplifying mouse ΔCtΔTmCD40L+ spacer using a mouse CD40ligand cDNA template are set forth below.

Mouse ΔCtΔTmCD40L+ spacer forward primer (MCD40LSPF): (SEQ ID NO: 50)5′-CCG CTCGAG AAC GAC GCA CAA GCA CCA AAA TCA AAG GTCGAAGAGGAAGTAAAC-3′;Mouse CD40L reverse primer (MCD40LR): (SEQ ID NO: 51) 5′-CCC TCTAGA ATCAGAGTTTCACTAAGCCAA-3′.

These primers will amplify a ΔCtΔTmCD40L+spacer which encodes 52-260 ofmouse CD40L. The forward primer MCD40LSPF encodes a 10 residue spacer(LENDAQAPKS) (SEQ ID NO:48) to be located between the antigen and theCD40 ligand (MCD40L) of the transcription unit. PCR was performed usingthe forward and reverse primers (SEQ ID NOs 50 and 51) and plasmidpDC406-mCD40L as the template under the following conditions: hold 3 minat 94° C.; cycle 94° C. for 45 sec, 55° C. for 45 sec, 72° C. for 70 sec(30 cycles); hold 7 min at 72° C.; and hold at 4° C. This PCR resultedin a fragment “spacer+ΔCtΔTmCD40L”which was subcloned into pcDNA3TOPO.The “spacer+ΔCtΔTmCD40L” fragment was then inserted into the plasmidpcDNA-sig-AfluHA after restriction endonuclease digestion with XbaI(TCTAGA) and Xho I (CTCGAG). The sig-AfluHA/ΔCtΔTmCD40L (mouse) encodingDNA was cut from the pCDNA3TOPO using HindIII-XbaI restriction andinserted into pShuttle-CMV (see Murphy et al., Prostate 38: 73-78, 1999)downstream of the CMV promoter. This vector is designated pShuttlesig-AfluHA/ΔCtΔTmCD40L(mouse). Thus, the transcription unitsig-AfluHA-ΔCtΔTmCD40L(mouse) encodes the kappa secretory signalfollowed by the extracellular domain of Avian flue HA followed by a 10amino acid linker (LI NDAQAPKS; SEQ ID NO:48) followed by mouse CD40ligand residues 52-260.

This amplified H5 HA sequence is cloned so that it is located between asecretory mouse Ig kappa sequence (sig) and a linker (sp), which linkerconnects to the gene encoding the ecdhCD40L (extracellular domain of thehuman CD40L) in an AdEasy shuttle vector. Vector and soluble fusionprotein expressed therefrom will be prepared as described above.

ii) H5HA-ecdCD40L(human)

The construction of a vector encoding an HA/human CD40 ligand fusionprotein (pShuttle-K/FIA-sp-ΔCtΔTmCD40L(human)) is described as follows.Primers for amplifying human ΔCtΔTMCD40L+ spacer using a human CD40ligand cDNA template are set forth below.

Human ΔCtΔTmCD40L+ spacer forward primer (HCD40LSPF) (CD40L sequenceitalicized): (SEQ ID NO: 66)

CAT AGA AGG TTG GAC-3′; Human CD40L reverse primer (HCD40LR)(SEQ ID NO: 67) 5′-CCC  TCTAGA  TCAGAGTTTGAGTAAGCCAAAGGAC-3′.

PCR is performed using the above primers and the plasmid pDC406-hCD40Las template under the following conditions: hold 3 min at 94° C.; cycle94° C. for 45 sec, 52° C. for 45 sec, 72° C. for 70 sec (30 cycles);hold 7 min at 72° C.; and hold at 4° C. This amplification results inthe “-sp-ΔCtΔTmCD40L(human)” fragment, which encodes 44-261 of humanCD40L and an amino terminal 10 aa spacer. The forward primer HCD40LSPFencodes a 10 residue spacer (LENDAQAPKS; single letter code; SEQ IDNO:48) to be located between the tumor antigen and the CD40 ligand(hCD40L) of the transcription unit. The “sp-ΔCtΔTmCD40L(human)” fragmentis subcloned into pcDNA3TOPO. The “spacer+ΔCtΔTmCD40L(human)” fragmentis then inserted into the plasmid pcDNA-sig-AfluHA after restrictionendonuclease digestion with XbaI (TCTAGA) and Xho I (CTCGAG). Thesig-AfluHA/ΔCtΔTmCD40L(human) encoding DNA is cut from the pCDNA3TOPOusing KpnI-XbaI restriction and inserted into pShuttle-CMV (see Murphyet al., Prostate 38: 73-78, 1999) downstream of the CMV promoter. Thisvector is designated pShuttle sig-AfluHA/ΔCtΔTmCD40L(human). Thus, thetranscription unit sig-AfluHA-ΔCtΔTmCD40L(human) encodes the kappasecretory signal followed by the extracellular domain of Avian flue HAfollowed by a 10 amino acid linker (LENDAQAPKS; SEQ ID NO:48) followedby human CD40 ligand residues 44-261.

This amplified H5 HA sequence is cloned so that it is located between asecretory mouse Ig kappa sequence (sig) and a linker (sp), which linkerconnects to the gene encoding the ecdhCD40L (extracellular domain of thehuman CD40L) in an AdEasy shuttle vector. Recombinant adenoviral vectorswere generated using the AdEasy vector system (Stratagene, San Diego,Calif.) as described above, resulting in the Ad-K/HA/ecdCD40L vector.Fusion protein were prepared as described below.

b) Induction_(.) of Immunity in Mice

The Ad-sig-H5HA/ecdhCD40L vector was processed for administration inaccordance with the methods of reference PNAS 2003 100:15101-15106;Blood 2004 104:2704-2713, was injected twice (at a 7 day interval) scinto immunocompetent mice (2 months old). Seven days following theimmunization, the CD8 cells were isolated, and incubated for 2 days inthe presence of the H5HA epitope (cultured with syngeneicgamma-irradiated spleen cells pulsed with HA peptides and cultured for48 hours, and developed according to the ELISPOT protocol (see PNAS 2003100:15101-15106; Blood 2004 104:2704-2713), and then the spleen cellswere tested by ELISPOT assay for the level of H5HA specific CD8 T cellsbefore and after vaccination. As shown in FIG. 18, the level of H5HAspecific CD8 T cells increased significantly in the spleen of thevaccinated mice (N=4) compared to the unvaccinated mice (N-4), p<0.05.

c) Induction of Immunity in Old and Young Mice

Two month old (“Young”) C57BL6 mice (n=4) and in 18 month old (“Old”)C57BL6 mice (n=4) were administered with one subcutaneous injection ofAd-sig-HA/ecdCD40L vector followed by 3 separate injections ofHA/ecdCD40L fusion protein at 7 day intervals starting 7 days after theinitial vector injection. Age-matched unvaccinated mice served ascontrols. The levels of HA specific splenic CD8 T cells were determinedby ELISPOT assay and the levels of the HA specific serum antibodies weredetermined by ELISA assay for all groups. As shown in FIG. 23, thelevels of HA specific splenic CD8 T cells of the vaccinated mice werestatistically significantly increased in the vaccinated groups ascompared to an age-matched unvaccinated control group for the 2 monthold mice (p=0.0004) and the 18 month old mice (p=0.0001). FIG. 24 showsthat the level of serum antibodies specific for the H5N1 HA antigen wasstatistically significantly increased in the vaccinated groups ascompared to an age-matched unvaccinated control group for 2 month oldmice (p=0.004) and 18 month old mice (p=0.015) (determined at a 1/250dilution).

15. Induction of Immunity with the Ad-sig-H5N1M2/ecdCD40L Vector Againstthe M2 Protein of the H5N1 Avian Influenza Virus in 2 Month Old Mice and18 Month Old Mice

The H5N1 M2 protein does not change in strains with influenza viralstrains harboring transitions of sequence in the HA antigens. However,it is a weak antigen during vaccination. Thus, a portion of the codingsequence of the M2 gene was linked to the extracellular domain of CD40ligand in the Ad-sig-M2/ecdCD40L to increase its immunogenicity.

a) Construction of Ad-sig-H5N1M2/ecdCD40L Vectors

As discussed, the M2 protein is not normally immunogenic, but can bemade so by including it in a chimeric protein with another immunogenicprotein sequence. The amino terminal end or an internal “immunodominantloop” from the extracellular domain of the M2 was used in such chimericstructures.

An Ad-K-M2/ecdCD40L vector where the M2 is from an H5N1 influenza virusis constructed to generate an immune response to the M2.

The amino acid sequence of a portion of an M2 protein corresponding toamino acid residues 1-24 of the M2 protein of an H5N1 Influenza A virus(A/Hong Kong/156/97), wherein the cysteine residues at positions 17 and19 in the native protein have been mutated to serine residues asreported by De Fillette et al. (Virology 337:149-61, 2005) is shownbelow.

(SEQ ID NO: 77) MSLLTEVDTLTRNGWGSRSSDSSD

Oligonucleotides corresponding to the sense and antisense strands of theDNA encoding SEQ ID NO:77 are as follows:

M2 sense oligonucleotide: (SEQ ID NO: 78) 5′-ATGA GCCTTCTAAC CGAGGTTGACACGCTTACCAGA AACGGATGGG GGTCCAGATC CAGCGATTCA AGTGAT-3′ M2 antisenseoligonucleotide: (SEQ ID NO: 79)5′-ATCACTTGAATCGCTGGATCTGGACCCCCATCCGTTTCTGGTAAGC GTGTCAACCTCGGTTAGAAGGCTCAT-3′

The oligonucleotides were dissolved in STE buffer at high concentration(about 1-10 OD₂₆₀ units/100 μL). The two strands were mixed together inequal molar amounts and heated to 94° C. and slowly cooled to about 4°C. The annealed DNA was phosphorylated using T4 PNK (polynucleotidekinase) under standard conditions. The resulting double stranded DNA wasamplified by PCR using the following primers:

Primer 1: (SEQ ID NO: 80) 5′-ACG ATG GAG ACA GAC ACA CTC CTG CTA TGG GTACTG CTG-3′ Primer 2 (SEQ ID NO: 81) 5′-TC CTG CTA TGG GTA CTG CTG CTCTGG GTT CCA GGT TC-3′ Primer 3: (SEQ ID NO: 82) 5′-TG CTC TGG GTT CCAGGT TCC ACT GGT GAC ATG-3′; Primer 4: (SEQ ID NO: 83) 5′-TGG GTT CCA GGTTCC ACT GGT GAC ATG ATGA GCCTTCTAAC CGAGGTTGAC-3′; and Primer 5: (SEQ IDNO: 84) 5′-CCG CTCGAG ATCACTTGAATCGCTGCATCTGCACC-3′.

The K/Avian FluM2 fragment having an upstream kappa signal sequence wasgenerated by four rounds of PCR amplification (1⁵ round: primers 4 +5;2^(nd) round: add primer 3; 3^(rd) round: add primer 2; 4^(th) round:add primer 1). The K/Avian FluM2 encoding DNA was subcloned into thepcDNA™ 3.1 TOPO vector (Invitrogen, San Diego, Calif.) resulting inplasmid pcDNA-K/Avian FluM2.

The “spacer+ΔCtΔTmCD40L(mouse)” fragment was cut from thepcDNA3TOPO-sp-ΔCtΔTmCD40L using Xho I and Xba I and was inserted intothe plasmid pcDNA-K/Avian FluM2 after restriction endonuclease digestionwith XbaI (TCTAGA) and Xho I (CTCGAG). The K/AvianFluM2/ΔCtΔTmCD40L(mouse) encoding DNA was cut from the pCDNA3TOPO using HindIII-XbaIrestriction and inserted into pShuttle-CMV downstream of the CMVpromoter. This vector is designated pShuttle sigAfluM2/ΔCtΔTmCD40L(mouse).

Recombinant adenoviral vectors were generated using the AdEasy vectorsystem (Stratagene, San Diego, Calif.) as described above, resulting inthe Ad-K/AvianFluM2/ecdCD40L vector. Fusion protein was prepared asdescribed below.

b) Induction of Immunity in Old and Young Mice

Two month old C57BL6 mice (n=5) and in 18 month old C57BL6 mice wereadministered with one subcutaneous injection of Ad-sig-M2/ecdCD40Lvector followed by injections of M2/ecdCD40L fusion protein 7 and 21days after the initial vector injection. Age-matched unvaccinated miceserved as controls. The levels of M2 specific splenic CD8 T cells weredetermined by ELISPOT assay and the levels of the M2 specific serumantibodies were determined by ELISA assay for all groups. As shown inFIG. 25, the levels of M2 specific splenic CD8 T cells of the vaccinatedmice were statistically significantly increased in the vaccinated groupsas compared to an age-matched unvaccinated control group for both the 2month old ( )0.0006), as well as the 18 month old mice (p=0.0009). FIG.26 shows that the level of serum antibodies specific for the H5N1 M2antigen was statistically significantly increased in the vaccinatedgroups as compared to an age-matched unvaccinated control group for 2month old mice (p=0.0028) and 18 month old mice (p=0.0025) (determinedat a 1/250 dilution). These data shows that the linkage of the CD40L tothe M2 protein in the Ad-sig-M2/ecdCD40L vaccine prime-M2/ecdCD40Lprotein boost vaccine induces a significant immune response.

16. Construction of Ad-sig-H5HA-M2/ecdCD40L chimeric vectors

An Ad-sig-TAA/ecdCD40L vectors carrying an antigen which is a chimericfusion of the HA and the M2 proteins is constructed as follows. The H5N1HA and M2 sequences are synthesized by PCR amplification from an H5N1template. An exemplary DNA sequence encoding an H5N1 HA antigen is asfollows.

(SEQ ID NO: 69) 5′- AAAAGTTCTTGGTCCAATCATGATGCCTCATCAGGGGTGAGCTCAGCATGTCCATACCTTGGGAGGTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTGCATACCCAACAGCTACTAGACCCAAAGTAAACGGGCAAAGTGGAAGAATGGAGTTCTTCTGGACAATTTTAAAG-3′.DNA sequence encoding a chimera HA-M2 is shown below with the H5N1 HAsequence 5′ to the M2 sequence. The underlined segment is added sequencebetween the above two segments.

(SEQ ID NO: 43) 5′-AAAAGTTCTTGGTCCAATCATGATGCCTCATCAGGGGTGAGCTCAGCATGTCCATACCTTGGGAGGTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTGCATACCCAACAGCTACTAGACCCAAAGTAAACGGGCAAAGTGGAAGAATGGAGTTCTTCTGGACAATTTTAAAGGATATCATGAGCCTTCTAACCGAGGTTGACACGCTTACCAGAAACGGATGGGGGTGCAGATGCAGCGATTCAAGTGAT-3′.The above DNA encodes the following chimeric HA-M2 protein:

(SEQ ID NO: 85) KSSWSNHDASSGVSSACPYLGRSSFFRNVVWLIKKNSAYPTATRPKVNGQSGRMEFFWTILKDIMSLLTEVDTLTRNGWGCRCSDSSD.

The above HA-M2 chimeric sequence is cloned downstream of a secretorysignal sequence (e.g., mouse 1gG kappa chain signal or secretorysequence) (sig). This sig HA-M2 molecule is then linked via a 30nucleotide linker sequence to DNA for ecdhCD40L. The TAA/ecdCD40Ltranscription unit is inserted into the Ad-sig-TAA/ecdCD40L vector bymethods previously described and reported (see e.g., PNAS 2003100:15101-15106; Blood 2004 104:2704-2713). The vector is then isolatedfrom 293 cells, plaque purified, and the TAA/ecdCD40L insert sequencedand tested for replication competent adenovirus by PCR assay for EIA.

17. Testing and use of the Adenovirus Expression Constructs

HA, NA, or M2 from H5N1, H3N2 or other Type A viral strains such as H2N2or H1N1 are cloned into the TAA/ecdCD40L vector. The influenza fusionprotein is expressed and used as a protein boost. Generally, the vectoris administered two times (7 days apart), or a vector is injectionfollowed by 2 protein boosts at 7 and 21 days. Subjects are vaccinatedwith the Ad-sig-TAA/ecdCD40L vector where the TAA is replaced with aninfluenza antigen.

a) In Vitro Stability of TAA/ccdCD40L and Activation of DCs

The stability of the expressed influenza antigen proteins is evaluatedas follows. The TAA/ecdCD40L protein is generated with a hexhis tag andonce released from 293 infected cells, it is purified by a nickelcolumn. The protein is studied by reducing and non-reducing SDS-PAGEgels to determine if the trimeric structure is stable with the HA, M2 orHAM2 as antigen. Western blotting is used with a flag antibody to testthe relative stability of the HA/ecdCD40L, M2/ecdCD40L and theHAM2/ecdCD40L proteins by methods reported previously (see Blood 2004104:2704-2713). The recombinant protein is added to mouse bone marrowderived dendritic cells (DCs) by methods reported previously (Deisserothet al., DC Vector Vaccine and Chemotherapy in Breast Cancer, Submitted2006). The endpoint of these studies is: FACS analysis of the DCs forappearance of CD 86 and PCR assay for expression of the CCR7 gene (PNAS2004 100:15101-15106; Blood 2004 104:2704-2713),

b) In Vivo Induction of Immune Response with each Ad-sig-TAA/ecdCD40L

Induction of immune response is evaluated for each influenza vaccinevector as follows, Two sc injections at 7 day intervals are administeredto 2 month and 18 month old BalbC mice. Seven, 14 and 21 days followingthe last injection, the animal is sacrificed and the following assayscarried out.

-   -   a. ELISA for AG specific antibodies in serum (see Blood 2004        104: 2704-2713 for method).    -   b. ELISPOT and CTL assays for level of AG specific CD8 effector        cells in spleen (see Blood 2004 104: 2704-2713 for method).    -   c. Determine the levels of CD8 Effector T cells and CD4 T        negative regulatory cells at the site of AGG inflammation.

The levels of CD8 and CD4 cells are evaluated. Two and 18 month old BACmice are injected sc with a syngeneic tumor cell line carrying the HA orM2 or HAM2 transcription unit. When the tumor nodule reaches the size of150 cu mm, the test mouse is injected sc with the Ad-sig-TAA/ecdCD4011,vector (2 injections 7 days apart). Seven days following the lastvaccination, the animal is sacrificed. The tumor is minced, treated withDNAase and collagenase, filtered through nylon mesh and the frequency ofCD8 effector cells and CD4CD25FOXP3 T neeative regulatory cells ismeasured by FACS before and after vaccination.

d) Suppression of Growth of TAA Positive Syngeneic Tumor Cell Line

Suppression of growth of influenza antigen positive cell-lines is alsoevaluated. The syngeneic HA positive or M2 positive or HAM2 positivesyngeneic cell line (500,000 cells) are injected sc into 2 month or 18month old mice. A growth curve is followed in mice that were vaccinatedwith two Ad-sig-TAA/ecdCD40L injections (7 days apart) prior to theinjection of the tumor cell line. The growth curve is the endpoint ofthe experiment.

e) Effect of Vaccination Based on One Ad-sig-TAA/ecdCD40L sc InjectionFollowed by Two TAAJecdCD40L Protein Injections as Boost

In the above Examples, it was shown that a single Ad-sig-TAA/ecdCD40L scinjection followed by two sc injections of 10 microgram of theTAA/ecdCD40L protein at 7 and 21 days produced dramatic increases in thelevels of both antigen specific antibodies in the serum and TAA specificCD8 effector T cells in test mice. Influenza antigen Ad-sig-TAA/ecdCD40Lsingle vector injection followed by two sc (10 microgram) TAAJecdCD40Lprotein boost injections at 7 and 21 days is used.

The number of boosts, the route of administration of the vaccine, andthe addition of adjuvant are modified to illicit an adequate increase inantigenic specific CD8 cells as determined by ELISPOT (200 positivecells/100,000 spleen cells) or antibodies (6-fold increase).

18. Induction of Immunity with Influenza Antigen/CD40 Ligand FusionProtein Against the Hemagglutinin Protein of an H5N1 Influenza A Virusin 2 Month Old Mice

SEQ ID NO:21 represents a portion of the HA sequence from an H5N1Influenza A virus (A/Hong Kong/156/97) was used as an antigen in theHA-CD40L fusion protein. As described below, this fusion protein inducedan immune response in mice; as shown by the level of anti-E15 HAantibody in the serum of vaccinated animals.

SEQ ID NO:21 is present in the receptor binding region corresponding toamino acid residues 135-175 connected to residues 230-250 (see FIG. 2,underlined) of the hemagglutinin (HA) protein of the H5N1 avianinfluenza strain (HSHA). This strain was first isolated in 1997 in HongKong from a child with a fatal respiratory illness (see, e.g., Science1998 279:393-96; J. Virology 1998 73: 2094-98; Emerging InfectiousDisease 2002 8(8):1-12).

(SEQ ID NO: 21) KSSWSNHDASSGVSSACPYLGRSSFFRNVVWLIKKNSAYPTATRPKVNGQSGRMEFFWTILK.

The HA-CD40L fusion was prepared as follows. A CD40L plasmid wasgenerated by PCR amplifying HSF1/ΔCtΔTmCD40L using plasmidpcDNA-sig-hMUC-1/HSF1/ΔCtΔTmCD40L as template (the construction of thisplasmid is described in U.S. Patent Application Publication US2005-0226888). The PCR primers and conditions for PCR are as follows:

Primer 1 (forward) (XcmI restriction site underlined; EcoRV restrictionsite underlined and italicized): (SEQ ID NO: 86) 5′-AACCA TCA CTC TTC TGG T GAGCTC  AAA GATATC AACGA CGCACAAGC-3′; Primer 2(forward) (EcoRV restriction site underlined and italicized): (SEQ IDNO: 87) 5′-GCTCAAA GATATC  AACGACGCACAAGCACCAAAATCA AAGGTC-3′; andPrimer 3 (reverse) (EcoRI restriction site underlined and bold): (SEQ IDNO: 88) 5′-AT CTCGAG CG GAATTC  CAGAGTTTGAGTAAGCCAAAAGA TGAGAAGCC-3′.HSF1/ΔCtΔTm CD40L was amplified by two rounds of PCR amplification (1stround: primers 2+3; 2nd round: primer 1+3). PCR was conducted using theGC-RICH PCR kit (Roche, Inc) under the following conditions:

Cycles Temperature Time 1 95° C. 3 min. 30 95° C. 45 sec. 55° C. 45 sec.72° C. 1 min. 1 72° C. 7 min.The HSF1/ΔCtΔTm CD40L encoding DNA was subcloned into pTriEx-2restriction sites XcinI (CCA TCA CTC TTC TGG) and EcoRt (GAATTC). Thefinal vector was named pTriEx-2 HSF1/ΔCtΔTm CD40L.

The cDNA encoding the HA antigen above (SEQ ID NO:21) was amplified fromplasmid pcDNA-K/Avian FluHA (described above) using the following PCRprimers:

HA forward primer (XcmI restriction site underlined): (SEQ ID NO: 89)5′-AA CCATCACTCTTCTGG TAGATCTAAAAGTTCTTGGTCCAATC-3′ and HA reverseprimer (XhoI restriction site underlined): (SEQ ID NO: 90) 5′-AAACTCGAG TCT GATATC CTTTAAAATTGTCCAGAAGAACTC-3′.PCR was conducted under the following conditions:

Cycles Temperature Time 1 95° C. 3 min. 30 95° C. 30 sec. 58° C. 30 sec.72° C. 30 sec. 1 72° C. 7 min.

The PCR amplification product was ligated into pcDNA3.1/V5-His TOPO TAvector and named as pcDNA3.1/HA then subcloned into the expressionplasmid pTriEx-2 HSF1/ΔCtΔTmCD40L using the XcmI and EcoRV sites toproduce the plasmid pTriEx-2HA/mCD40L. Competent cells (Rosetta DE3pLace were transformed with pTriEx-2HAmCD40L and single colony oftransformed cells was selected. His-tagged HA/mCD40L fusion protein wasproduced from the transformed cells using the Overnight Express™Autoinduction System (Novagen, Following protein expression, theresulting media containing the cells was centrifuged at 5000 g for 10minutes and the resulting supernatant discarded. Cells were lysed usingthe CelLytic B Plus Kit (Sigma, Inc.) as follows: 10 mL Lytic Buffer wasadded for each gram of pelleted cells; the Lytic Buffer was prepared byadding 10 mL CelLytic B reagent, 0.2 mL lysozyme, 0.1 mL proteaseinhibitor and 500 U benzonase; cells were resuspended in Lytic Bufferand incubated with shaking at RT for 10-15 minutes; and lysed cells werecentrifuged 16,000g for 10 minutes. Fusion protein was purified byHIS-Select Nickel Affinity Gel (Sigma, Inc.) as follows: 1 mL ofHis-Select Nickel Affinity Gel was prepared for each 10 mL of celllysate supernatant and the fusion protein isolated using the “BatchMethod” protocol from the manufacturer and performing 3 wash stepsbefore the final elution of the His-tagged fusion protein; and theeluted protein (in 3 mL buffer) was applied to a 10DG desalting columnand extracted from the column using 4 mL PBS. The eluted protein wasconcentrated using a Vivaspin column.

The H5HA/ecdCD40L fusion protein (10 μg) was injected subcutaneouslyinto immunocompetent mice (2 months old) on days 0, 7, and 21 with orwithout 100 μg aluminum hydroxide hydrogel. Seven days following thethird immunization, serum samples were collected and assayed by ELISAfor antibody to HA. As shown in FIG. 25, the level of H5HA specificantibodies in both groups (i.e., with and without aluminum hydroxide)are significantly higher in the sera of vaccinated mice (N=4) comparedto the control mice injected only with PBS (N=4).

The neutralizing dose (ND₅₀) of the antibody to HA was determined usingthe following method.

MDCK cells were harvested and the cell suspension diluted to 3×10⁵cells/mL (or other appropriate concentration so that the cells are atleast 90% confluent at the initiation of theassay). 200 μL of thediluted cell suspension was transferred into each well of a 96-wellmicroplate and incubated at 37±2° C. and 5.0±2% CO₂ for a minimum of 16hours in a humidified incubator until the wells were at least 90%confluent. Serum samples were obtained from the above-described micevaccinated with HA/ecdCD40L, 7 days after the third immunization. 300 μLaliquots of serum were serially diluted in inoculation medium (dilutionsof 1:5 to 1:2560). The virus challenge solution was prepared by dilutingthe challenge virus to 200 TCID₅₀/100 μl (or 2×10³ TCID₅₀/mL) ininoculation media. 300 μL of virus challenge solution was added to eachserum dilution to produce inoculating solutions having a final serumdilution of 1:10 to 1:5120 and incubated at 37±2° C. and 5.0±2% CO₂ in ahumidified incubator for 1 hour±5 minutes. Each well of the platecontaining the MDCK cells was washed once with 200 μL Hank's BalancedSalt Solution (HBSS), inoculated with 100 μL of inoculating solution andincubated at 37±2° C. and 5.0±2% CO₂ in a humidified incubator for aminimum of 16 hours or until cytopathological effects (CPE) are evidentin comparison to control (no virus) wells. The ND₅₀ is the dilution thatresults in the absence of CPE in 50% of the wells inoculated wascalculated using the Spearman Kärber method.

At a targeted challenge concentration of virus of 20 (TC₅₀/mL), theserum from HA immunized animals showed a neutralizing titer of 4.56×10³.At a ten-fold higher dilution of challenge virus (i.e., 200 (TC₅₀/mL))no neutralizing effect of the serum from HA immunized animals wasobserved.

19. Induction of Immunity with Influenza Antigen/CD40 Ligand FusionProtein Against the M2 Protein of an HSM Influenza A virus in 2 MonthOld Mice

A portion of the M2 amino acid sequence from an H5N1 Influenza A virus(A/Hong Kong/156/97) was used as an antigen in the M2/ecdCD40L fusionprotein. As described below, this fusion protein induced an immuneresponse in mice; that is, a significant level of anti-M2 antibody wasdetected in the serum of vaccinated animals.

The amino acid sequence of a portion of an M2 protein corresponding toamino acid residues 1-24 of the M2 protein of an H5N1 Influenza A virus(A/Hong Kong/156/97), wherein the cysteine residues at positions 17 and19 in the native protein have been mutated to serine residues asreported by De Fillette et al. (Virology 337:149-61, 2005) is shownbelow.

(SEQ ID NO: 77) MSLLTEVDTLTRNGWGSRSSDSSD

Oligonucleotides corresponding to the sense and antisense strands of theDNA encoding SEQ ID NO:77 are as follows:

M2 sense oligonucleotide: (SEQ ID NO: 78) 5′-ATGA GCCTTCTAAC CGAGGTTGACACGCTTACCAGA AACGGATGGG GGTCCAGATC CAGCGATTCA AGTGAT-3′ M2 antisenseoligonucleotide: (SEQ ID NO: 79)5′-ATCACTTGAATCGCTGGATCTGGACCCCCATCCGTTTCTGGTAAGC GTGTCAACCTCGGTTAGAAGGCTCAT-3′The oligonucleotides were dissolved in STE buffer at high concentration(about 1-10 OD₂₆₀ units/100 μL). The two strands were mixed together inequal molar amounts and heated to 94° C. and slowly cooled to about 4°C. The annealed DNA was phosphorylated using T4 PNK (polynucleotidekinase) under standard conditions. The resulting double stranded DNA wasamplified by PCR using the following primers:

M2 forward primer (XcmI restriction site underlined): (SEQ ID NO: 91)5′-AA CCA TCA CTC TTC TGG T AGATCT ATGA GCCTTCTAAC CGAGGTTGAC-3 M2reverse primer (XhoI restriction site underlined): (SEQ ID NO: 92)5′-AAA CTCGAG TCT GATATC ATCACTTGAATCGCTGGATCTGG-3′PCR was conducted under the following conditions:

Cycles Temperature Time 1 95° C. 3 min. 30 95° C. 30 sec. 58° C. 30 sec.72° C. 20 sec. 1 72° C. 7 min.

The PCR amplification product was ligated into pcDNA3.1/V5-His TOPO TAvector and named as pcDNA3.1/M2 then subcloned into the expressionplasmid pTriEx-2 HSF1/ΔCtΔTmCD40L using the XcmI and EcoRV sites toproduce the plasmid pTriEx-2M2/mCD40L. Competent cells (Rosetta DE3pLacI) were transformed with pTriEx-2 M2mCD40L and single colony oftransformed cells was selected. His-tagged M2/mCD40L fusion protein wasproduced from the transformed cells using the Overnight Express™Autoinduction System (Novagen, Inc.). Following protein expression, theresulting media containing the cells was centrifuged at 5000 g for 10minutes and the resulting supernatant discarded. Cells were lysed usingthe CelLytic B Plus Kit (Sigma, Inc.) as follows: 10 mL Lytic Buffer wasadded for each gram of pelleted cells; the Lytic Buffer was prepared byadding 10 mL CelLytic B reagent, 0.2 mL lysozyme, 0.1 mL proteaseinhibitor and 500 U benzonase, cells were resuspended in Lytic Bufferand incubated with shaking at RT for 10-15 minutes; and lysed cells werecentrifuged 16,000 g for 10 minutes. The fusion protein was purified byHIS-Select Nickel Affinity Gel (Sigma, Inc.) as follows: 1 mL ofHis-Select Nickel Affinity Gel was prepared for each 10 mL of celllysate supernatant and the fusion protein isolated using the “BatchMethod” protocol from the manufacturer and performing 3 wash stepsbefore the final elution of the His-tagged fusion protein; and theeluted protein (in 3 mL buffer) was applied to a 10DG desalting columnand extracted from the column using 4 mL PBS. The eluted protein wasconcentrated using a Vivaspin column.

The M2/ecdCD40L fusion protein (10 μg) was injected subcutaneously intoimmunocompetent mice (2 months old) on days 0, 7, and 21 with or without100 μg aluminum hydroxide hydrogel. Seven days following the thirdimmunization, serum samples were collected and assayed by ELISA forantibody to M2. As shown in FIG. 26, the level of M2 specific antibodiesin both groups (i.e., with and without aluminum hydroxide) aresignificantly higher in the sera of vaccinated mice (N=4) compared tothe control mice injected only with PBS (N=4).

The neutralizing dose (ND₅₀) of the sera from M2 immunized mice wasdetermined using the method as described in the preceding example. At atargeted challenge concentration of virus of 20 (TC₅₀/mL), the serumfrom M2 immunized animals showed a neutralizing titer of 1.27×10². At aten-fold higher dilution of challenge virus (i.e., 200 (TC₅₀/mL)) noneutralizing effect of the serum from M2 immunized animals was observed.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A vaccine composition for generating in anindividual an immune response against one or more influenza antigensresulting from an infectious influenza virus, comprising: an effectiveamount of an expression vector, said expression vector comprising atranscription unit encoding a secretable fusion protein comprising atleast first and second influenza antigen fragments, each influenzaantigen fragment from an extracellular region of the influenza virus,and each of said first and second influenza antigen fragments comprisingat least first and second epitopes wherein the first and secondinfluenza antigen fragments are linked to the amino terminus of anextracellular domain of a CD40 ligand, for generating both a cellularand antibody immune response, said first influenza antigen fragmentcomprising, the first epitope that is recognized and bound by Class IIMHC for eliciting antigen specific antibodies, and the second epitopethat is recognized and bound by Class I MHC for eliciting antigenspecific CD8⁺ effector T cells, and said second influenza antigenfragment comprising the first epitope that is recognized and bound byClass II MHC for eliciting antigen specific antibodies, and the secondepitope that is recognized and bound by Class I MHC for elicitingantigen specific CD8⁺ effector T cells, wherein each of said epitopes inboth the first and second influenza antigen fragments has a distinctamino acid sequence from each of the other epitopes.
 2. The compositionof claim 1 wherein said influenza antigen fragments are from aninfluenza virus belonging to at least one or both of classes H3N2 orH5N1.
 3. The composition of claim 1 wherein each of said influenzaantigen fragments is from a different viral influenza protein.
 4. Thecomposition of claim 1 wherein at least one of said first and secondinfluenza antigen fragments is from the extracellular domain ofhemagglutinin or neuraminidase or Matrix protein
 2. 5. The compositionof claim 1 wherein said expression vector is a viral vector or a plasmidvector.
 6. The composition of claim 5 wherein said viral vector is anadenoviral vector.
 7. The composition of claim 1 wherein said antibodiesare neutralizing antibodies.
 8. The composition of claim 1 wherein thetranscription unit encodes a secretory signal sequence linked to thefirst and second influenza antigen fragments connected to a linkerconnected to the extracellular domain of the CD40 ligand.
 9. A vaccinecomposition for increasing the immune responsiveness in an individualagainst one or more foreign antigens on an infectious agent, comprising:an expression vector comprising a transcription unit encoding asecretable fusion protein comprising two or more foreign antigenfragments each from the extracellular domain of the one or more foreignantigens on the infectious agent, and an extracellular domain of a CD40Lligand, wherein the two or more foreign antigen fragments are linked toan amino terminus of the extracellular domain of the CD40L ligand, afirst one of said foreign antigen fragments comprising a first epitopethat contains a binding site that is recognized and bound by Class IIMHC for eliciting foreign antigen specific antibodies, and furthercomprising a second epitope that contains a binding site that isrecognized and bound by Class I MHC for eliciting foreign antigenspecific CD8⁺ T cells, and a second one of said foreign antigenfragments comprising a first epitope and a second epitope, each of saidfirst and second epitopes in the second foreign antigen fragment havinga distinct amino acid sequence from each of the amino acid sequenceepitopes in the first foreign antigen fragment, and said first andsecond epitopes of said second foreign antigen each having a bindingsite respectively recognized and bound by Class II MHC and Class I MHC.10. The composition of claim 9 wherein said infectious agent comprisesone or more proteins of the same strain or different strains and saidforeign antigen fragments are from said one or more proteins.
 11. Thecomposition of claim 9 wherein said infectious agent is one ofbacterial, fungal, viral or protozoan.
 12. The composition of claim 9wherein said foreign antigen specific antibodies are neutralizingantibodies.
 13. A vaccine composition employing an expression vectorcomprising a transcription unit encoding a secretable fusion proteincomprising first and second connected and distinct foreign antigenfragments, each fragment from a different protein of an infectiousagent, and an extracellular domain of a CD40 ligand, said first foreignantigen fragment from a first protein comprising at least first andsecond epitopes linked to the amino terminus of the extracellular domainof said CD40 ligand, wherein: the first one of said epitopes thatcontains antigenic determinant that includes a binding site that isrecognized and bound by Class II MHC for eliciting foreign antigenspecific antibodies, and the second one of said epitopes that containsantigenic determinant that includes a binding site that is recognizedand bound by Class I MHC for eliciting foreign antigen specificCD8⁺effector T cells, and said second foreign antigen fragment from asecond protein, and comprising at least a first epitope and a secondepitope, said second fragment first epitope and second epitope eachcontaining a binding site that is respectively recognized and bound byClass II MHC and Class I MHC.
 14. The composition of claim 13 whereinsaid foreign antigen specific antibodies are neutralizing antibodies.15. The composition of claim 13 wherein the first and second foreignantigen fragments are each from proteins of the same strain or differentstrains.
 16. The composition of claim 13, wherein said foreign antigenfragments are one of bacterial, fungal, viral or protozoan.
 17. Acomposition for generating an immune response in an individual againstone or more influenza antigens on an infectious agent for limitingprogression of the infectious agent for multiple strains of theinfluenza virus, comprising, an expression vector comprising atranscription unit encoding a secretable fusion protein comprising theextracellular domain of the CD40 ligand and an extracellular portion ofthe Matrix protein 2 influenza antigen SEQ ID NO 77, wherein said Matrixprotein 2 is connected at the amino terminus of the CD40 ligand andfunctions to increase the immunogenicity of said Matrix protein 2 so asto increase both the level of Matrix protein 2 specific CD8⁺ effector Tcells and neutralizing antibodies, against the infectious agent.